Human antibodies to pd-l1

ABSTRACT

The present invention provides antibodies that bind to the T-cell co-inhibitor ligand programmed death-ligand1 (PD-L1) protein, and methods of use. In various embodiments of the invention, the antibodies are fully human antibodies that bind to PD-L1. In certain embodiments, the present invention provides multi-specific antigen-binding molecules comprising a first binding specificity that binds to PD-L1 and a second binding specificity that binds to a tumor cell antigen, an infected cell-specific antigen, or a T-cell co-inhibitor. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing PD-L1 activity, thus providing a means of treating a disease or disorder such as cancer or viral infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/905,653 filed Feb. 26, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/603,808 filed Jan. 23, 2015 (now U.S. Pat. No.9,938,345), which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/930,582 filed Jan. 23,2014 and U.S. Provisional Application No. 62/089,549 filed Dec. 9, 2014,the disclosures of all of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind to theimmunomodulatory receptor ligand programmed death-ligand 1 (PD-L1), andtherapeutic and diagnostic methods of using those antibodies.

STATEMENT OF RELATED ART

Programmed death-ligand 1 (PD-L1) (also called B7-H1 or CD274) is a 290amino acid protein receptor ligand expressed widely on both lymphoid andnon-lymphoid tissues such as CD4 and CD8 T-cells, macrophage lineagecells, peripheral tissues as well as on tumor cells, andvirally-infected cells (Dong et al 1999, Nature Med.). PD-L1 binds toreceptors PD-1 and B7-1 which belong to the CD28/CTLA-4 (cytotoxic Tlymphocyte antigen)/ICOS (inducible co-stimulator) family of T-cellco-inhibitory receptors (Chen et al 2013, Nature Rev. Immunol. 13:227-242) and attenuates the immune response by inhibiting T-cellactivation. PD-L1 binding to PD-1 or B7-1 results in decreased T-cellproliferation and cytokine secretion, compromising humoral and cellularimmune responses in diseases such as cancer, and viral infection.

The expression of PD-L1 on tumor cells and virally-infected cells isexploited by tumors and chronic viral infections to evade immuneresponse. PD-L1 is expressed on a wide variety of tumors and studies onanimal models have shown that PD-L1 on tumors inhibits T-cell activationand lysis of tumor cells and may lead to increased death oftumor-specific T-cells. In chronic viral infections, PD-L1 expressed onvirally-infected cells binds to PD-1 on virus-specific T-cells and theseT-cells become “exhausted” with loss of effector functions andproliferative capacity (Freeman 2008, PNAS 105: 10275-10276). The PD-1:PD-L1 system also plays an important role in induced T-regulatory (Treg)cell development and in sustaining Treg function (Francisco et al 2010,Immunol. Rev. 236: 219-242).

Since PD-L1 plays an important role in tumor immunity and infectiousimmunity, it is an ideal target for immunotherapy. Blocking PD-L1 withantagonists, including monoclonal antibodies, has been studied intreatments of cancer and chronic viral infections (Ribas 2012, NEJM 366:2517-2519; Freeman 2008, PNAS 105: 10275-10276; Sheridan 2012, NatureBiotechnology 30: 729-730).

Monoclonal antibodies to PD-L1 are known in the art and have beendescribed, for example, in US Patent/Publication Nos. 7943742, 8383796,8217149, 20090055944, 20120003056, 20130034559, 20130045200,20130045201, 20130045202, and in WO2007005874, WO2011066389,WO2010077634, EP1907424, and EP1899379.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that bind PD-L1. The antibodies of the present invention areuseful, inter alia, for targeting cells expressing PD-L1 such as cancercells or virally-infected cells, and for modulating PD-L1 activity. Incertain embodiments, the antibodies of the invention are useful forinhibiting or neutralizing PD-L1 activity and for stimulating T cellactivation, e.g., under circumstances where T cell-mediated killing isbeneficial or desirable. The anti-PD-L1 antibodies of the invention, orantigen-binding portions thereof, may be included as part of amulti-specific antigen-binding molecule, for example, to modulate theimmune response and/or to target the antibodies to a specific cell type,such as a tumor cell, or a virally infected cell. The antibodies areuseful in treating a disease or disorder such as cancer and viralinfection.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933). In certain embodiments, theantibodies may be bispecific.

In a first aspect, the present invention provides isolated monoclonalantibodies or antigen-binding fragments thereof that bind specificallyto PD-L1. Exemplary anti-PD-L1 antibodies of the present invention arelisted in Tables 1 and 2 herein. Table 1 sets forth the amino acidsequence identifiers of the heavy chain variable regions (HCVRs), lightchain variable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-PD-L1antibodies. Table 2 sets forth the nucleic acid sequence identifiers ofthe HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of theexemplary anti-PD-L1 antibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-PD-L1 antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is selected from the group consistingof SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/170, 186/194, 202/210, 218/226, 234/242,250/258, 266/274, 282/274, 290/274, 298/274, 306/274, 314/274, 322/274,330/274, and 338/274. In certain embodiments, the HCVR/LCVR amino acidsequence pair is selected from one of SEQ ID NOs: 82/90 (e.g.,H2M8314N), 162/170 (e.g., H2M8718N), 306/274 (e.g., H1H9364P2), and314/274 (e.g., H1H9373P2). In certain other embodiments, the HCVR/LCVRamino acid sequence pair is selected from one of SEQ ID NOs: 98/106(e.g., H2M8316N), 146/154 (e.g., H2M8323N), 290/274 (e.g., H1H9351P2),and 330/274 (e.g., H1H9387P2).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-PD-L1 antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is selected fromthe group consisting of SEQ ID NOs: 88/96 (e.g., H2M8314N), 168/176(e.g., H2M8718N), 312/280 (e.g., H1H9364P2), and 320/280 (e.g.,H1H9373P2). In certain other embodiments, the HCDR3/LCDR3 amino acidsequence pair is selected from the group consisting of SEQ ID NOs:104/112 (e.g., H2M8316N), 152/160 (e.g., H2M8323N), 296/280 (e.g.,H1H9351P2), and 336/280 (e.g., H1H9387P2).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-PD-L1 antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequenceset is selected from the group consisting of SEQ ID NOs:84-86-88-92-94-96 (e.g., H2M8314N); 164-166-168-172-174-176 (e.g.,H2M8718N); 308-310-312-276-278-280 (e.g., H1H9364P2); and316-318-320-276-278-280 (e.g., H1H9373P2). In certain other embodiments,the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set isselected from the group consisting of SEQ ID NOs:100-102-104-108-110-112 (e.g., H2M8316N); 148-150-152-156-158-160 (e.g.,H2M8323N); 292-294-296-276-278-280 (e.g., H1H9351P2); and332-334-336-276-278-280 (e.g., H1H9387P2).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary anti-PD-L1antibodies listed in Table 1. For example, the present inventionincludes antibodies, or antigen-binding fragments thereof, comprisingthe HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences setcontained within an HCVR/LCVR amino acid sequence pair selected from thegroup consisting of SEQ ID NOs: 82/90 (e.g., H2M8314N), 98/106 (e.g.,H2M8316N), 146/154 (e.g., H2M8323N), 162/170 (e.g., H2M8718N), 290/274(e.g., H1H9351P2), 306/274 (e.g., H1H9364P2), 314/274 (e.g., H1H9373P2)and 330/274 (e.g., H1H9387P2). Methods and techniques for identifyingCDRs within HCVR and LCVR amino acid sequences are well known in the artand can be used to identify CDRs within the specified HCVR and/or LCVRamino acid sequences disclosed herein. Exemplary conventions that can beused to identify the boundaries of CDRs include, e.g., the Kabatdefinition, the Chothia definition, and the AbM definition. In generalterms, the Kabat definition is based on sequence variability, theChothia definition is based on the location of the structural loopregions, and the AbM definition is a compromise between the Kabat andChothia approaches. See, e.g., Kabat, “Sequences of Proteins ofImmunological Interest,” National Institutes of Health, Bethesda, Md.(1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martinet al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databasesare also available for identifying CDR sequences within an antibody.

The present invention includes anti-PD-L1 antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

The present invention also provides for antibodies and antigen-bindingfragments thereof that compete for specific binding to PD-L1 with anantibody or antigen-binding fragment thereof comprising the CDRs of aHCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an aminoacid sequence selected from the HCVR and LCVR sequences listed in Table1.

The present invention also provides isolated antibodies andantigen-binding fragments thereof that block PD-L1 binding to PD-1 or toB7-1. In some embodiments, the antibody or antigen-binding fragmentthereof that blocks PD-L1 binding to PD-1 or to B7-1 may bind to thesame epitope on PD-L1 as PD-1/B7-1 or may bind to a different epitope onPD-L1 as PD-1/B7-1. In certain embodiments, the antibodies of theinvention that block PD-L1 binding to PD-1 or to B7-1 comprise the CDRsof an HCVR having an amino acid sequence selected from the groupconsisting of HCVR sequences listed in Table 1; and the CDRs of a LCVRhaving an amino acid sequence selected from the group consisting of LCVRsequences listed in Table 1.

In alternate embodiments, the present invention provides antibodies andantigen-binding fragments thereof that do not block PD-L1 binding toPD-1 or to B7-1. In certain embodiments, the present invention providesisolated antibodies or antigen-binding fragments thereof that bindPD-L1, wherein the antibodies or antigen-binding fragments thereofenhance PD-L1 binding to PD-1 or to B7-1. In some embodiments, theisolated antibodies or antigen-binding fragments thereof that enhancePD-L1 binding to PD-1/B7-1 comprise the CDRs of a HCVR, wherein the HCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 18, 66, 114, 130, 202, 218, 266, 282, 298, 322 and 338; and theCDRs of a LCVR, wherein the LCVR has an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 26, 74, 122, 138, 210, 226, and274. In some embodiments, the isolated antibodies or antigen-bindingfragments thereof comprise an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 18/26 (e.g.,H2M8307N), 66/74 (e.g., H2M8312N), 114/122 (e.g., H2M8317N), 130/138(e.g., H2M8321N), 202/210 (e.g., H1H9323P), 218/226 (e.g., H1H9327P),266/274 (e.g., H1H9344P2), 282/274 (e.g., H1H9345P2), 298/274 (e.g.,H1H9354P2), 322/274 (e.g., H1H9382P2), and 338/274 (e.g., H1H9396P2).

The present invention also provides antibodies and antigen-bindingfragments thereof that bind specifically to PD-L1 from human or otherspecies. In certain embodiments, the antibodies may bind to human PD-L1and/or to cynomolgus PD-L1.

The present invention also provides antibodies and antigen-bindingfragments thereof that cross-compete for binding to PD-L1 with areference antibody or antigen-binding fragment thereof comprising theCDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR eachhas an amino acid sequence selected from the HCVR and LCVR sequenceslisted in Table 1.

In one embodiment, the invention provides an isolated antibody orantigen-binding fragment that has one or more of the followingcharacteristics: (a) blocks the binding of PD-L1 to PD-1 or to B7-1; (b)binds specifically to human PD-L1 and/or cynomolgus PD-L1; (c) inhibitsT-cell proliferation in a mixed lymphocyte reaction (MLR) assay; and (d)increases IL-2 and/or interferon-gamma secretion in a MLR assay.

In some embodiments, the antibody or antigen binding fragment thereofmay bind specifically to PD-L1 in an agonist manner, i.e., it mayenhance or stimulate PD-L1 binding and/or activity; in otherembodiments, the antibody may bind specifically to PD-L1 in anantagonist manner, i.e., it may block PD-L1 from binding to itsreceptor.

In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention are bispecific comprising a first bindingspecificity to PD-L1 and a second binding specificity for a secondtarget epitope. The second target epitope may be another epitope onPD-L1 or on a different protein such as a T-cell co-inhibitor. Incertain embodiments, the target epitope may be on a different cellincluding e.g., a different T-cell, a B-cell, a tumor cell, anautoimmune tissue cell or a virally infected cell.

In a second aspect, the present invention provides nucleic acidmolecules encoding anti-PD-L1 antibodies or portions thereof. Forexample, the present invention provides nucleic acid molecules encodingany of the HCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PD-L1 antibodies listedin Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-PD-L1 antibodies listedin Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-PD-L1 antibody listed in Table1.

In a related aspect, the present invention provides recombinantexpression vectors capable of expressing a polypeptide comprising aheavy or light chain variable region of an anti-PD-L1 antibody. Forexample, the present invention includes recombinant expression vectorscomprising any of the nucleic acid molecules mentioned above, i.e.,nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDRsequences as set forth in Table 1. Also included within the scope of thepresent invention are host cells into which such vectors have beenintroduced, as well as methods of producing the antibodies or portionsthereof by culturing the host cells under conditions permittingproduction of the antibodies or antibody fragments, and recovering theantibodies and antibody fragments so produced.

In a third aspect, the present invention provides multi-specificantigen-binding molecules and antigen-binding fragments thereofcomprising a first antigen-binding specificity that binds specificallyto PD-L1 and a second antigen-binding specificity that bindsspecifically to an antigen selected from the group consisting of PD-L1,a tumor cell-specific antigen, an infected-cell-specific antigen, and aT-cell co-inhibitor. In certain embodiments, the first antigen-bindingspecificity may comprise three CDRs derived from a HCVR with an aminoacid sequence selected from the HCVR sequences in Table 1 and three CDRsderived from a LCVR with an amino acid sequence selected from the LCVRsequences in Table 1. In one embodiment, the first antigen-bindingspecificity may comprise an extracellular domain of PD-1 or of B7-1, ora fragment thereof. The second antigen-binding specificity may target anantigen on the same cell as PD-L1 or on a different cell of the sametissue type or of a different tissue type. For example, themulti-specific antigen-binding molecule may bind to a T-cell wherein thefirst antigen-binding specificity may bind specifically to PD-L1 and thesecond antigen-binding specificity may bind to a T-cell co-inhibitor onthe T-cell. Alternatively, in another embodiment, the firstantigen-binding specificity binds specifically to PD-L1 on a T-cell andthe second antigen-binding specificity is targeted to anantigen/receptor on a B-cell or a macrophage or antigen-presenting cell.In certain embodiments, the second antigen-binding specificity isdirected to an antigen on a tumor cell, or on a cell infected with avirus. In one embodiment, the first antigen-binding specificitycomprises an extracellular domain of PD-1 and the second antigen-bindingspecificity binds to a different epitope on PD-L1. In certainembodiments, the first antigen-binding specificity binds to PD-L1 with alower affinity, for example, with a K_(D) more than 10⁻⁸ M, more than10⁻⁷ M, more than 10⁻⁶ M, or more than 10⁻⁵ M.

In a fourth aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or antigen-binding fragmentthereof which specifically binds PD-L1 and a pharmaceutically acceptablecarrier. In a related aspect, the invention features a composition whichis a combination of an anti-PD-L1 antibody and a second therapeuticagent. In one embodiment, the second therapeutic agent is any agent thatis advantageously combined with an anti-PD-L1 antibody. Exemplary agentsthat may be advantageously combined with an anti-PD-L1 antibody include,without limitation, other agents that bind and/or modulate PD-L1signaling (including other antibodies or antigen-binding fragmentsthereof, etc.) and/or agents which do not directly bind PD-L1 butnonetheless modulate immune cell activation. Additional combinationtherapies and co-formulations involving the anti-PD-L1 antibodies andmulti-specific antigen-binding molecules of the present invention aredisclosed elsewhere herein.

In a fifth aspect, the invention provides methods to modulate the immuneresponse in a subject, the method comprising administering atherapeutically effective amount of an anti-PD-L1 antibody orantigen-binding fragment thereof of the invention to the subject in needthereof. In certain embodiments, the invention provides methods toenhance the immune response in a subject, the methods comprisingadministering to the subject an effective amount of an antibody orfragment thereof of the invention that binds PD-L1 and blocks PD-L1binding to PD-1 or to B7-1. In one embodiment, the invention provides amethod to stimulate or enhance T-cell activation in a subject, themethod comprising administering a blocking antibody or antigen-bindingfragment thereof of the invention to the subject in need thereof. In oneembodiment, the invention provides methods to inhibit a T-regulatory(Treg) cell in a subject, the methods comprising administering ablocking antibody or antigen-binding fragment thereof of the inventionto the subject in need thereof. In certain embodiments, the subject inneed thereof may suffer from a disease or disorder such as cancer orviral infection. In alternate embodiments, the present inventionprovides methods to inhibit T-cell activation, the methods comprisingadministering an activating antibody or antigen-binding fragment thereofof the invention to a subject in need thereof. In certain furtherembodiments, the subject in need thereof may suffer from an autoimmunedisease.

In a sixth aspect, the invention provides therapeutic methods fortreating a disease or disorder, for example, cancer or viral infection,in a subject using an anti-PD-L1 antibody or antigen-binding portion ofan antibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or fragment of an antibody of theinvention to the subject in need thereof. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by stimulation or inhibition of PD-L1 binding or activity. Incertain embodiments, the antibody or antigen-binding fragment thereofthe invention is administered in combination with a second therapeuticagent to the subject in need thereof. The second therapeutic agent maybe selected from the group consisting of an antibody to a T-cellco-inhibitor, an antibody to a tumor cell antigen, an antibody to aT-cell receptor, an antibody to a Fc receptor, an antibody to an epitopeon a virally infected cell, an antibody to PD-1, a cytotoxic agent, ananti-cancer drug, an anti-viral drug, an anti-inflammatory drug (e.g.,corticosteroids), a VEGF antagonist, and any other drug or therapy knownin the art. In certain embodiments, the second therapeutic agent may bean agent that helps to counteract or reduce any possible side effect(s)associated with an antibody or antigen-binding fragment thereof of theinvention, if such side effect(s) should occur.

In certain embodiments, the present invention provides methods forsuppressing tumor growth. In certain embodiments, the present inventionprovides methods to enhance survival of cancer patients. Examples ofcancer include, but are not limited to, primary and/or recurrent cancer,including brain cancer (e.g., glioblastoma multiforme), lung cancer(e.g., non-small cell lung cancer), squamous cell carcinoma of head andneck, renal cell carcinoma, melanoma, multiple myeloma, prostate cancer,and colon cancer. The methods comprise administering a pharmaceuticalcomposition comprising a therapeutically effective amount of ananti-PD-L1 antibody of the present invention in combination with asecond therapeutic agent selected from the group consisting of avascular endothelial growth factor (VEGF) antagonist (e.g., aflibercept,bevacizumab), an angiopoietin-2 (Ang2) inhibitor (e.g., an anti-Ang2antibody such as nesvacumab), a lymphocyte activation gene 3 (LAG-3)inhibitor, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor (e.g.,ipilimumab), a CCR4 inhibitor (e.g., mogamulizumab), a chemotherapeuticagent, and radiation therapy. Additional examples of additionaltherapies/therapeutic agents that can be used in combination with ananti-PD-L1 antibody of the invention for use in treating cancer aredescribed elsewhere herein.

The antibody or fragment thereof may be administered subcutaneously,intravenously, intradermally, intraperitoneally, orally,intramuscularly, or intracranially. The antibody or fragment thereof maybe administered at a dose of about 0.1 mg/kg of body weight to about 100mg/kg of body weight of the subject.

The present invention also includes use of an anti-PD-L1 antibody orantigen-binding fragment thereof of the invention in the manufacture ofa medicament for the treatment of a disease or disorder (e.g., cancer,and chronic viral infection) that would benefit from the blockade orenhancement of PD-L1 binding and/or signaling.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of the luciferase-based PD-L1 bioassay describedin Example 8 herein. Panel A: Inactive Jurkat cells; Panel B: Jurkatcells are activated by T-cell receptor (TCR) clustering through theCD3×CD20 bispecific antibody; Panel C: PD-1 activation attenuatesresponse in activated Jurkat cells; Panel D: Blocking PD-L1 rescues theresponse in activated Jurkat cells.

FIG. 2 illustrates tumor growth and survival results for mice implantedwith Colon-26 tumor cells at Day 0 and treated with the indicatedcombinations of molecules by injection at Days 3, 6, 10, 13 and 19(“early-treatment tumor model”). The graph depicts tumor volume (in mm³)for the different experimental groups at various time points afterimplantation. Upward arrows along the X-axis indicate the timing oftreatment injections. “mlgG2a” is IgG2 isotype control; “Fc” is human Fccontrol; “VEGF Trap” is aflibercept; “anti-PD-1” is anti-mouse PD-1clone RPMI-14; “anti-PD-L1” is an anti-PD-L1 monoclonal antibody asdescribed elsewhere herein.

FIG. 3 illustrates tumor growth and survival results for mice implantedwith Colon-26 tumor cells at Day 0 and treated with the indicatedcombinations of molecules by injection at Days 3, 6, 10, 13 and 19(“early-treatment tumor model”). The graph shows the tumor volume (inmm³) of individual mice in each experimental group at Day 28 afterimplantation. “mlgG2a” is IgG2 isotype control; “Fc” is human Fccontrol; “VEGF Trap” is aflibercept; “anti-PD-1” is anti-mouse PD-1clone RPMI-14; “anti-PD-L1” is an anti-PD-L1 monoclonal antibody asdescribed elsewhere herein.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The term “PD-L1” refers to programmed death-ligand 1, also known asCD274 and B7H1. The amino acid sequence of full-length PD-L1 is providedin GenBank as accession number NP 054862.1 and is also referred toherein as SEQ ID NO: 351. The term “PD-L1” also includes proteinvariants of PD-L1 having the amino acid sequence of SEQ ID NOs: 345,346, 347 or 348. The term “PD-L1” includes recombinant PD-L1 or afragment thereof. The term also encompasses PD-L1 or a fragment thereofcoupled to, for example, histidine tag, mouse or human Fc, or a signalsequence such as ROR1. For example, the term includes sequencesexemplified by SEQ ID NOs: 347 or 348, comprising a mouse Fc (mlgG2a) orhuman Fc (hIgG1) at the C-terminal, coupled to amino acid residues19-239 of full-length PD-L1 (SEQ ID NO: 351; NP_054862.1). Proteinvariants as exemplified by SEQ ID NO: 345 comprise a histidine tag atthe C-terminal, coupled to amino acid residues 19-239 of NP_054862.1.Unless specified as being from a non-human species, the term “PD-L1”means human PD-L1.

PD-L1 is a 290 amino acid protein with extracellular IgV-like andIgC-like domains (amino acids 19-239 of full length PD-L1), atransmembrane domain and an intracellular domain of approximately 30amino acids. PD-L1 is constitutively expressed on many cells such asantigen presenting cells (e.g., dendritic cells, macrophages, andB-cells) and on hematopoietic and non-hematopoietic cells (e.g.,vascular endothelial cells, pancreatic islets, and sites of immuneprivilege). PD-L1 is also expressed on a wide variety of tumors, andvirally-infected cells and is a component of the immunosuppressivemilieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of twoT-cell co-inhibitors PD-1 and B7-1.

The term “PD-1” refers to the programmed death-1 protein, a T-cellco-inhibitor, also known as CD279. The amino acid sequence offull-length PD-1 is provided in GenBank as accession number NP 005009.2and is also referred to herein as SEQ ID NO: 352. The term alsoencompasses PD-1 or a fragment thereof coupled to, for example,histidine tag, mouse or human Fc, or a signal sequence such as ROR1. Forexample, the term includes sequences exemplified by SEQ ID NOs: 349 or350, comprising a mouse Fc (mlgG2a) or human Fc (hIgG1) at theC-terminal, coupled to amino acid residues 25-170 of NP_005009.2 with aC93S change.

PD-1 is a member of the CD28/CTLA-4/ICOS family of T-cell co-inhibitors.PD-1 is a 288-amino acid protein with an extracellular N-terminal domainwhich is IgV-like, a transmembrane domain and an intracellular domaincontaining an immunoreceptor tyrosine-based inhibitory (ITIM) motif andan immunoreceptor tyrosine-based switch (ITSM) motif (Chattopadhyay etal 2009, Immunol. Rev.). The PD-1 receptor has two ligands, PD-L1 andPD-L2.

The term “B7-1” refers to the T-lymphocyte activation antigen, alsoknown as costimulatory factor CD80. B7-1 is a 288 amino acid membranereceptor with an extracellular N-terminal domain which comprisesIgV-like (aa 37-138) and IgC-like (aa 154-232) regions, a transmembranedomain (aa 243-263) and a C-terminal intracellular region (aa 263-288).The amino acid sequence of full-length B7-1 is provided in GenBank asaccession number NP_005182.1.

As used herein, the term “T-cell co-inhibitor” refers to a ligand and/orreceptor which modulates the immune response via T-cell activation orsuppression. The term “T-cell co-inhibitor”, also known as T-cellco-signaling molecule, includes, but is not limited to, PD-1, lymphocyteactivation gene 3 protein (LAG-3, also known as CD223), cytotoxicT-lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA),CD-28, 2B4, LY108, T-cell immunoglobulin and mucin-3 (TIM3), T-cellimmunoreceptor with immunoglobulin and ITIM (TIGIT; also known asVSIG9), leucocyte associated immunoglobulin-like receptor 1 (LAIR1; alsoknown as CD305), inducible T-cell costimulator (ICOS; also known asCD278), B7-1 (CD80), and CD160.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (CL). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully human anti-PD-L1 monoclonal antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes fully human anti-PD-L1 monoclonalantibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-PD-L1antibodies having HCVR, LCVR, and/or CDR amino acid sequences with,e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservativeamino acid substitutions relative to any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences. The term includes antibodiesrecombinantly produced in a non-human mammal, or in cells of a non-humanmammal. The term is not intended to include antibodies isolated from orgenerated in a human subject.

The term “multi-specific antigen-binding molecules”, as used hereinrefers to bispecific, tri-specific or multi-specific antigen-bindingmolecules, and antigen-binding fragments thereof. Multi-specificantigen-binding molecules may be specific for different epitopes of onetarget polypeptide or may contain antigen-binding domains specific forepitopes of more than one target polypeptide. A multi-specificantigen-binding molecule can be a single multifunctional polypeptide, orit can be a multimeric complex of two or more polypeptides that arecovalently or non-covalently associated with one another. The term“multi-specific antigen-binding molecules” includes antibodies of thepresent invention that may be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein. For example, anantibody or fragment thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, non-covalent association orotherwise) to one or more other molecular entities, such as a protein orfragment thereof to produce a bi-specific or a multi-specificantigen-binding molecule with a second binding specificity. According tothe present invention, the term “multi-specific antigen-bindingmolecules” also includes bi-specific, tri-specific or multi-specificantibodies or antigen-binding fragments thereof. In certain embodiments,an antibody of the present invention is functionally linked to anotherantibody or antigen-binding fragment thereof to produce a bispecificantibody with a second binding specificity. Bispecific andmulti-specific antibodies of the present invention are describedelsewhere herein.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁸ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically toPD-L1. Moreover, multi-specific antibodies that bind to one domain inPD-L1 and one or more additional antigens or a bi-specific that binds totwo different regions of PD-L1 are nonetheless considered antibodiesthat “specifically bind”, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to PD-L1, expressed as K_(D), of at least 10⁻⁸ M; preferably10⁻⁹ M; more preferably 10⁻¹⁰ M, even more preferably 10⁻¹¹ M, even morepreferably 10⁻¹² M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from PD-L1, with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind to PD-L1.

In specific embodiments, antibody or antibody fragments of the inventionmay be conjugated to a moiety such a ligand or a therapeutic moiety(“immunoconjugate”), such as an antibiotic, a second anti-PD-L1antibody, or an antibody to another antigen such a tumor-specificantigen, a virally-infected cell antigen, or a T-cell co-inhibitor, oran immunotoxin, or any other therapeutic moiety useful for treating adisease or condition including e.g., cancer, chronic viral infection orautoimmune disease.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds PD-L1, or a fragment thereof, is substantially freeof Abs that specifically bind antigens other than PD-L1.

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes PD-L1 activity” or an “antagonistantibody”), is intended to refer to an antibody whose binding to PD-L1results in inhibition of at least one biological activity of PD-L1. Forexample, an antibody of the invention may prevent or block PD-L1 bindingto PD-1 or to B7-1.

An “activating antibody” or an “enhancing antibody”, as used herein (oran “agonist antibody”), is intended to refer to an antibody whosebinding to PD-L1 results in increasing or stimulating at least onebiological activity of PD-L1. For example, an antibody of the inventionmay increase or enhance PD-L1 binding to PD-1 or to B7-1.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, the term “subject” refers to an animal, preferably amammal, in need of amelioration, prevention and/or treatment of adisease or disorder such as chronic viral infection, cancer orautoimmune disease.

As used herein, “anti-cancer drug” means any agent useful to treatcancer including, but not limited to, cytotoxins and agents such asantimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P′-(DDD)), interferons and radioactiveagents. As used herein, “a cytotoxin or cytotoxic agent” means any agentthat is detrimental to cells. Examples include Taxol® (paclitaxel),cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

As used herein, the term “anti-viral drug” refers to any drug or therapyused to treat, prevent, or ameliorate a viral infection in a hostsubject. The term “anti-viral drug” includes, but is not limited tozidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz,cobicistat, tenofovir, rilpivirine, analgesics and corticosteroids. Inthe context of the present invention, the viral infections includelong-term or chronic infections caused by viruses including, but notlimited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV),hepatitis C virus (HCV), human papilloma virus (HPV), lymphocyticchoriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).

The antibodies and antigen-binding fragments of the present inventionspecifically bind to PD-L1 and modulate the interaction of PD-L1 withPD-1 or with B7-1. The anti-PD-L1 antibodies may bind to PD-L1 with highaffinity or with low affinity. In certain embodiments, the antibodies ofthe present invention are blocking antibodies wherein the antibodiesbind to PD-L1 and block the interaction of PD-L1 with PD-1 or with B7-1.In some embodiments, the blocking antibodies of the invention block thebinding of PD-L1 to PD-1 or to B7-1 and/or stimulate or enhance T-cellactivation. In some embodiments, the blocking antibodies are useful forstimulating or enhancing the immune response and/or for treating asubject suffering from cancer, or a chronic viral infection. Theantibodies when administered to a subject in need thereof may reduce thechronic infection by a virus such as HIV, LCMV or HBV in the subject.They may be used to inhibit the growth of tumor cells in a subject. Theymay be used alone or as adjunct therapy with other therapeutic moietiesor modalities known in the art for treating cancer, or viral infection.In certain embodiments, the anti-PD-L1 antibodies that bind to PD-L1with a low affinity are used as multi-specific antigen-binding moleculeswherein the first binding specificity binds to PD-L1 with a low affinityand the second binding specificity binds to an antigen selected from thegroup consisting of a different epitope of PD-L1, a T-cell co-inhibitorsuch as PD-1, a tumor specific antigen and an infected-cell-specificantigen.

In certain embodiments, the antibodies of the present invention areagonist antibodies, wherein the antibodies bind to PD-L1 and enhance theinteraction of PD-L1 and PD-1/B7-1. In some embodiments, the activatingantibodies enhance binding of PD-L1 to PD-1 or to B7-1 and/or inhibit orsuppress T-cell activation. The activating antibodies of the presentinvention may be useful for inhibiting the immune response in a subjectand/or for treating autoimmune disease.

In certain embodiments, the anti-PD-L1 antibodies are multi-specificantigen-binding molecules, wherein they comprise a first bindingspecificity to PD-L1 and a second binding specificity to an antigenselected from the group consisting of a different epitope of PD-L1, aT-cell co-inhibitor such as PD-1, a tumor specific antigen and aninfected-cell-specific antigen. In certain embodiments, the firstbinding specificity binds to PD-L1 with low affinity, e.g., with a K_(D)of 10⁻⁸ M, 10⁻⁷ M or more.

Certain anti-PD-L1 antibodies of the present invention are able to bindto and neutralize the activity of PD-L1, as determined by in vitro or invivo assays. The ability of the antibodies of the invention to bind toand neutralize the activity of PD-L1 may be measured using any standardmethod known to those skilled in the art, including binding assays, oractivity assays, as described herein.

Non-limiting, exemplary in vitro assays for measuring binding activityare illustrated in Example 3, herein. In Example 3, the bindingaffinities and kinetic constants of human anti-PD-L1 antibodies forhuman PD-L1 and cynomolgus PD-L1 were determined by surface plasmonresonance and the measurements were conducted on a T200 Biacoreinstrument. In Examples 4 and 5, blocking assays were used to determinethe ability of the anti-PD-L1 antibodies to block PD-L1-binding abilityof PD-1 or to B7-1 in vitro. In Example 6, blocking assays were used todetermine cross-competition between different anti-PD-L1 antibodies.Example 7 describes the binding of the antibodies to cellsoverexpressing PD-L1. In Example 8, a luciferase assay was used todetermine the ability of anti-PD-L1 antibodies to antagonize PD-1/PD-L1signaling in T-cells.

In certain embodiments, the antibodies of the present invention are ableto enhance or stimulate T-cell activation in vitro and in a subject withcancer or in a subject infected with a virus such as LCMV. In certainembodiments, the antibodies of the present invention are used incombination with a second therapeutic agent, such as an antibody to atumor-specific antigen or a T-cell co-inhibitor, to enhance the immuneresponse and inhibit tumor growth in a subject. In certain embodiments,the agonist antibodies of the invention able to enhance PD-L1 binding toPD-1 or to B7-1 and may inhibit T-cell activation in vitro and/or in asubject with autoimmune disease.

The antibodies specific for PD-L1 may contain no additional labels ormoieties, or they may contain an N-terminal or C-terminal label ormoiety. In one embodiment, the label or moiety is biotin. In a bindingassay, the location of a label (if any) may determine the orientation ofthe peptide relative to the surface upon which the peptide is bound. Forexample, if a surface is coated with avidin, a peptide containing anN-terminal biotin will be oriented such that the C-terminal portion ofthe peptide will be distal to the surface. In one embodiment, the labelmay be a radionuclide, a fluorescent dye or a MRI-detectable label. Incertain embodiments, such labeled antibodies may be used in diagnosticassays including imaging assays.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding fragment”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to PD-L1. An antibody fragment may include a Fab fragment, aF(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing aCDR, or an isolated CDR. In certain embodiments, the term“antigen-binding fragment” refers to a polypeptide or fragment thereofof a multi-specific antigen-binding molecule. In such embodiments, theterm “antigen-binding fragment” includes, e.g., an extracellular domainof PD-1 which binds specifically to PD-L1. Antigen-binding fragments ofan antibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-antibodylibraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)—C_(H)1; (ii)V_(H)—C_(H)2; (iii) V_(H)—C_(H)3; (iv) V_(H)—C_(H)1-C_(H)2; (v)V_(H)—C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)—C_(H)2-C_(H)3, V_(H)—CL,V_(L)—C_(H)1; (ix) V_(L)-C_(H)2, (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3;V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-CL. In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind toPD-L1.

An immunogen comprising any one of the following can be used to generateantibodies to PD-L1. In certain embodiments, the antibodies of theinvention are obtained from mice immunized with a primary immunogen,such as a full length PD-L1 [See GenBank accession number NP_054862.1(SEQ ID NO: 351)] or with a recombinant form of PD-L1 or modified humanPD-L1 fragments (SEQ ID NOs: 345, 347 or 348) or with modifiedcynomolgus PD-L1 fragments (SEQ ID NO: 346), followed by immunizationwith a secondary immunogen, or with an immunogenically active fragmentof PD-L1.

In certain embodiments, the immunogen may be a peptide from the Nterminal or C terminal end of PD-L1. In one embodiment, the immunogen isthe extracellular IgV-like and/or IgC-like domain of PD-L1. In certainembodiments of the invention, the immunogen is a fragment of PD-L1 thatranges from about amino acid residues 19-239 of SEQ ID NO: 351(NP_054862.1).

In some embodiments, the immunogen may be a recombinant PD-L1 peptideexpressed in E. coli or in any other eukaryotic or mammalian cells suchas Chinese hamster ovary (CHO) cells.

In certain embodiments, antibodies that bind specifically to PD-L1 maybe prepared using fragments of the above-noted regions, or peptides thatextend beyond the designated regions by about 5 to about 20 amino acidresidues from either, or both, the N or C terminal ends of the regionsdescribed herein. In certain embodiments, any combination of theabove-noted regions or fragments thereof may be used in the preparationof PD-L1 specific antibodies.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to PD-L1 are initially isolated having a human variableregion and a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Bioequivalents

The anti-PD-L1 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies, but that retain the ability to bindPD-L1. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, theantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an antibody or antibody fragment that is essentiallybioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single dose or multipledoses. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,or potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include antibody variants comprising aminoacid changes, which modify the glycosylation characteristics of theantibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-PD-L1 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-PD-L1antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-PD-L1 antibodies comprising a mutationin the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W,H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). Inyet another embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-PD-L1 antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). In one embodiment, thepresent invention includes anti-PD-L1 antibodies comprising an Fc domaincomprising a S108P mutation in the hinge region of IgG4 to promote dimerstabilization. All possible combinations of the foregoing Fc domainmutations, and other mutations within the antibody variable domainsdisclosed herein, are contemplated within the scope of the presentinvention.

The present invention also includes anti-PD-L1 antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Ser. No. 14/170,166, filedJan. 31, 2014, the disclosure of which is hereby incorporated byreference in its entirety).

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention function by bindingto PD-L1. The present invention includes anti-PD-L1 antibodies andantigen-binding fragments thereof that bind soluble monomeric or dimericPD-L1 molecules with high affinity. For example, the present inventionincludes antibodies and antigen-binding fragments of antibodies thatbind monomeric PD-L1 (e.g., at 25° C. or at 37° C.) with a K_(D) of lessthan about 318 pM as measured by surface plasmon resonance, e.g., usingthe assay format as defined in Example 3 herein. In certain embodiments,the antibodies or antigen-binding fragments thereof bind monomeric PD-L1with a K_(D) of less than about 300 pM, less than about 250 pM, lessthan about 150 pM, less than about 100 pM, or less than about 50 pM, asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind dimeric PD-L1 (e.g., at 25° C. or at 37° C.)with a K_(D) of less than about 15 pM as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein.In certain embodiments, the antibodies or antigen-binding fragmentsthereof bind dimeric PD-L1 with a K_(D) of less than about 12 pM, lessthan about 10 pM, less than about 8 pM, or less than about 5 pM, asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies or antigen-bindingfragments thereof that bind cynomolgus (Macaca fascicularis) PD-L1(e.g., at 25° C. or at 37° C.) with a K_(D) of less than about 28 nM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 3 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof bind cynomolgus PD-L1 with a K_(D) ofless than about 25 nM, less than about 20 nM, less than about 15 nM,less than about 10 nM, or less than about 5 nM, as measured by surfaceplasmon resonance, e.g., using the assay format as defined in Example 3herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind PD-L1 with a dissociative half-life (t½) ofgreater than about 1 minute as measured by surface plasmon resonance at25° C. or 37° C., e.g., using an assay format as defined in Example 3herein, or a substantially similar assay. In certain embodiments, theantibodies or antigen-binding fragments of the present invention bindPD-L1 with a t % of greater than about 5 minutes, greater than about 10minutes, greater than about 30 minutes, greater than about 50 minutes,greater than about 60 minutes, greater than about 70 minutes, greaterthan about 80 minutes, greater than about 90 minutes, greater than about100 minutes, greater than about 200 minutes, greater than about 300minutes, greater than about 400 minutes, greater than about 500 minutes,greater than about 600 minutes, greater than about 700 minutes, orgreater than about 800 minutes, as measured by surface plasmon resonanceat 25° C. or 37° C., e.g., using an assay format as defined in Example 3herein (e.g., mAb-capture or antigen-capture format), or a substantiallysimilar assay.

The present invention also includes antibodies or antigen-bindingfragments thereof that block PD-L1 binding to PD-1 with an IC₅₀ of lessthan about 770 pM as determined using a ELISA-based immunoassay assay,e.g., as shown in Example 4, or a substantially similar assay. Thepresent invention also includes antibodies or antigen-binding fragmentsthereof that block PD-L1 binding to B7-1 with an IC₅₀ of less than about10 nM as determined using a ELISA-based immunoassay assay, e.g., asshown in Example 4, or a substantially similar assay. The presentinvention also includes antibodies and antigen-binding fragments thereofthat bind to PD-L1 and enhance the binding of PD-L1 to PD-1 or to B7-1.

In some embodiments, the antibodies of the present invention may bind tothe extracellular domain of PD-L1 or to a fragment of the domain. Insome embodiments, the antibodies of the present invention may bind tomore than one domain (cross-reactive antibodies). In certainembodiments, the antibodies of the present invention may bind to anepitope located in the extracellular domain comprising amino acidresidues 19-239 of NP_054862.1 (SEQ ID NO: 351). In some embodiments,the antibodies may bind to an epitope comprising one or more amino acidsselected from the group consisting of amino acid residues 1-221 of SEQID NOs: 345-348, or 353.

In certain embodiments, the antibodies of the present invention mayfunction by blocking or inhibiting the PD-1-binding or the B7-1-bindingactivity associated with PD-L1 by binding to any other region orfragment of the full length protein, the amino acid sequence of which isshown in SEQ ID NO: 351. In certain embodiments, the antibodies mayattenuate or modulate the interaction between PD-L1 and PD-1/B7-1.

In certain embodiments, the antibodies of the present invention may bebi-specific antibodies. The bi-specific antibodies of the invention maybind one epitope in one domain and may also bind a second epitope in adifferent domain of PD-L1. In certain embodiments, the bi-specificantibodies of the invention may bind two different epitopes in the samedomain. In one embodiment, the multi-specific antigen-binding moleculecomprises a first antigen-binding specificity wherein the first bindingspecificity comprises the extracellular domain or fragment thereof ofPD-1; and a second antigen-binding specificity to another epitope ofPD-L1. In another embodiment, the multi-specific antigen-bindingmolecule comprises a first antigen-binding specificity wherein the firstbinding specificity comprises the extracellular domain or fragmentthereof of B7-1; and a second antigen-binding specificity to anotherepitope of PD-L1.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that binds to PD-L1,wherein the antibody or fragment thereof exhibits one or more of thefollowing characteristics: (i) comprises a HCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130, 146, 162, 178, 186, 202, 218, 234, 250, 258, 266,274, 282, 290, 298, 306, 314, 322, 330 and 338, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (ii) comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 74, 90, 106, 122, 138, 154, 170, 194, 210, 226, 242, 258, and274, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; (iii)comprises a HCDR3 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136,152, 168, 184, 192, 208, 224, 240, 256, 272, 280, 288, 296, 304, 312,320, 328, 336 and 344, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a LCDR3 domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128,144, 160, 176, 200, 216, 232, 248, 264, and 280, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (iv) comprises a HCDR1 domain havingan amino acid sequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 188, 204, 220, 236,252, 268, 284, 292, 300, 308, 316, 324, 332, and 340, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a HCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54,70, 86, 102, 118, 134, 150, 166, 182, 190, 206, 222, 238, 254, 270, 286,294, 302, 310, 318, 326, 334, and 342, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a LCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 196, 212, 228, 244, 260, and 276,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a LCDR2domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 198, 214,230, 246, 262, and 278, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; (v) is a multi-specific antigen-binding molecule comprising afirst binding specificity to PD-L1 and a second binding specificity toan antigen selected from the group consisting of PD-L1, a tumor specificantigen, a virally infected cell antigen, and a T-cell co-inhibitor;(vi) binds to human PD-L1 with a K_(D) of about 4 pM to about 645 nM;(vii) binds to cynomolgus PD-L1 with a K_(D) of about 70 pM to about 400nM; (viii) blocks or enhances the binding of PD-L1 to PD-1 with anIC₅₀≤770 pM; (ix) blocks or enhances the binding of PD-L1 to B7-1 withan IC50≤10 nM; (x) blocks PD-1-induced T-cell down-regulation and/orrescues T-cell signaling in a T-cell/APC luciferase reporter assay; (xi)stimulates T-cell proliferation and activity in a mixed lymphocytereaction (MLR) assay; (xii) induces IL-2 and/or IFNγ production in a MLRassay; and (xiii) suppresses tumor growth and increases survival insubjects with cancer.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that blocks PD-L1 bindingto PD-1 or to B7-1, wherein the antibody or fragment thereof exhibitsone or more of the following characteristics: (i) comprises a HCVRhaving an amino acid sequence selected from the group consisting of SEQID NO: 82, 98, 146, 162, 290, 306, 314, and 330, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (ii) comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 90, 106,154, 170, and 274, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;(iii) comprises a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 88, 104, 152, 168, 296, 312,320, and 336, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a LCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 96, 112, 160, 176, and 280, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (iv) comprises a HCDR1 domain havingan amino acid sequence selected from the group consisting of SEQ ID NO:84, 100, 148, 164, 292, 308, 316, and 332, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a HCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 86, 102, 150,166, 294, 310, 318, and 334, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a LCDR1 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 92, 108, 156, 172, and 276, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR2 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 94, 110, 158, 174, and 278, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; (v) is a multi-specific antigen-binding moleculecomprising a first binding specificity to PD-L1 and a second bindingspecificity to an antigen selected from the group consisting of adifferent epitope of PD-L1, a tumor specific antigen, a virally-infectedcell antigen, and a T-cell co-inhibitor; (vi) binds to human PD-L1 witha K_(D)≤10⁻¹⁰M; (vii) binds to cynomolgus PD-L1 with a K_(D)≤10⁻⁷M;(viii) blocks the binding of PD-L1 to PD-1 or to B7-1; (ix) blocksPD-1-induced T-cell down-regulation and/or rescues T-cell signaling in aT-cell/APC luciferase reporter assay; (xi) stimulates T-cellproliferation and activity in a mixed lymphocyte reaction (MLR) assay;(xii) induces IL-2 and/or IFNγ production in a MLR assay; and (xiii)suppresses tumor growth and increases survival in subjects with cancer.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-PD-L1antibodies bind to human PD-L1 but not to PD-L1 from other species.Alternatively, the anti-PD-L1 antibodies of the invention, in certainembodiments, bind to human PD-L1 and to PD-L1 from one or more non-humanspecies. For example, the anti-PD-L1 antibodies of the invention maybind to human PD-L1 and may bind or not bind, as the case may be, to oneor more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog,rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus orchimpanzee PD-L1. In certain embodiments, the anti-PD-L1 antibodies ofthe invention may bind to human and cynomolgus PD-L1 with the sameaffinities or with different affinities.

Epitope Mapping and Related Technologies

The present invention includes anti-PD-L1 antibodies which interact withone or more amino acids found within one or more domains of the PD-L1molecule including, e.g., extracellular (IgV-like) domain, theextracellular IgC-like domain, a transmembrane domain, and anintracellular domain. The epitope to which the antibodies bind mayconsist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidslocated within any of the aforementioned domains of the PD-L1 molecule(e.g. a linear epitope in a domain). Alternatively, the epitope mayconsist of a plurality of non-contiguous amino acids (or amino acidsequences) located within either or both of the aforementioned domainsof the PD-L1 molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,for example, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Other methods include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (seeUS 2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theantibodies of the invention into groups of antibodies binding differentepitopes.

In certain embodiments, the anti-PD-L1 antibodies or antigen-bindingfragments thereof bind an epitope within any one or more of the regionsexemplified in PD-L1, either in natural form, as exemplified in SEQ IDNO: 351, or recombinantly produced, as exemplified in SEQ ID NOS:345-348, or to a fragment thereof. In some embodiments, the antibodiesof the invention bind to an extracellular region comprising one or moreamino acids selected from the group consisting of amino acid residues19-239 of PD-L1. In some embodiments, the antibodies of the inventionbind to a region comprising one or more amino acids selected from thegroup consisting of amino acid residues 1-221 of cynomolgus PD-L1, asexemplified by SEQ ID NO: 346.

In certain embodiments, the antibodies of the invention, as shown inTable 1, interact with at least one amino acid sequence selected fromthe group consisting of amino acid residues ranging from about position19 to about position 130 of SEQ ID NO: 351; or amino acid residuesranging from about position 130 to about position 153 of SEQ ID NO: 351;or amino acid residues ranging from about position 153 to about position210 of SEQ ID NO: 351; or to amino acid residues ranging from aboutposition 210 to about position 239 of SEQ ID NO: 351. These regions arepartially exemplified in SEQ ID NOs: 345-348.

The present invention includes anti-PD-L1 antibodies that bind to thesame epitope, or a portion of the epitope, as any of the specificexemplary antibodies described herein in Table 1, or an antibody havingthe CDR sequences of any of the exemplary antibodies described inTable 1. Likewise, the present invention also includes anti-PD-L1antibodies that compete for binding to PD-L1 or a PD-L1 fragment withany of the specific exemplary antibodies described herein in Table 1, oran antibody having the CDR sequences of any of the exemplary antibodiesdescribed in Table 1. For example, the present invention includesanti-PD-L1 antibodies that cross-compete for binding to PD-L1 with oneor more antibodies as defined in Example 6 herein (e.g., H2aM8309N,H1H9329P, H1H9336P, H2aM8314N, H2aM8316N, H2AM8718N, H1H9387P2,H1H9351P2, H1H9364P2, H1H9373P2, and H2aM8306N). The present inventionalso includes anti-PD-L1 antibodies that cross-compete for binding toPD-L1 with one or more antibodies as defined in Example 6 herein (e.g.,H1H9396P2, H2aM8317N, H2aM8321N, H1H9323P, H1H9382P2, H1H9344P2,H1H9345P2 and H1H9354P2).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PD-L1 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-PD-L1antibody of the invention, the reference antibody is allowed to bind toa PD-L1 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the PD-L1 molecule is assessed. Ifthe test antibody is able to bind to PD-L1 following saturation bindingwith the reference anti-PD-L1 antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-PD-L1antibody. On the other hand, if the test antibody is not able to bind tothe PD-L1 protein following saturation binding with the referenceanti-PD-L1 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-PD-L1 antibody of theinvention.

To determine if an antibody competes for binding with a referenceanti-PD-L1 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a PD-L1 protein under saturatingconditions followed by assessment of binding of the test antibody to thePD-L1 molecule. In a second orientation, the test antibody is allowed tobind to a PD-L1 molecule under saturating conditions followed byassessment of binding of the reference antibody to the PD-L1 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the PD-L1 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to PD-L1.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-PD-L1 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin or a chemotherapeutic agent to treat cancer. As used herein,the term “immunoconjugate” refers to an antibody which is chemically orbiologically linked to a cytotoxin, a radioactive agent, a cytokine, aninterferon, a target or reporter moiety, an enzyme, a toxin, a peptideor protein or a therapeutic agent. The antibody may be linked to thecytotoxin, radioactive agent, cytokine, interferon, target or reportermoiety, enzyme, toxin, peptide or therapeutic agent at any locationalong the molecule so long as it is able to bind its target. Examples ofimmunoconjugates include antibody drug conjugates and antibody-toxinfusion proteins. In one embodiment, the agent may be a second differentantibody to PD-L1. In certain embodiments, the antibody may beconjugated to an agent specific for a tumor cell or a virally infectedcell. The type of therapeutic moiety that may be conjugated to theanti-PD-L1 antibody and will take into account the condition to betreated and the desired therapeutic effect to be achieved. Examples ofsuitable agents for forming immunoconjugates are known in the art; seefor example, WO 05/103081.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244.

In one aspect, the present invention includes multi-specificantigen-binding molecules or antigen-binding fragments thereof whereinone antigen-binding specificity of an immunoglobulin is specific for anepitope within the extracellular domain of PD-L1 (e.g., in the IgV-likeregion), or a fragment thereof, and the other antigen-bindingspecificity of the immunoglobulin is specific for binding to a differentepitope in the extracellular domain of PD-L1 (e.g., in the IgC-likeregion), or a second therapeutic target, or is conjugated to atherapeutic moiety. In certain embodiments, the first antigen-bindingspecificity may comprise PD-1 or B7-1 or a fragment thereof. In oneembodiment, the first antigen-binding specificity that binds to PD-L1comprises the extracellular domain of PD-1. In certain embodiments ofthe invention, one antigen-binding specificity of an immunoglobulin isspecific for an epitope within amino acid residues 19-239 of PD-L1 (SEQID NO: 351) or a fragment thereof, and the other specificity of theimmunoglobulin is specific for a second target antigen. The secondtarget antigen may be on the same cell as PD-L1 or on a different cell.In one embodiment, the second target cell is on an immune cell otherthan a T-cell such as a B-cell, antigen-presenting cell, monocyte,macrophage, or dendritic cell. In some embodiments, the second targetantigen may be present on a tumor cell or on a virally infected cell.

In another aspect, the invention provides multi-specific antigen-bindingmolecules or antigen-binding fragments thereof comprising a firstantigen-binding specificity that binds to PD-L1 and a secondantigen-binding specificity that binds specifically to a target antigenon a tumor cell. In various embodiments, the tumor-specific antigen isone of CA9, CA125, melanoma-associated antigen (MAGE), carcinoembryonicantigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA),MART-1, or CA19-9. Non-limiting examples of other specifictumor-associated antigens include, e.g., AFP, ALK, BAGE proteins,β-catenin, brc-abl, BRCA1, BORIS, carbonic anhydrase IX, caspase-8,CCR5, CD19, CD20, CD30, CD40, CDK4, CTLA4, cyclin-B1, CYP1B1, EGFR,EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1,FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3,GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGEproteins (e.g., MAGE-1, -2,-3, -4, -6, and -12), HLA-A2, MART-1,mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1,NA17, NY-BR1, NY-BR62, NY-BR85, NY-ES01, OX40, p15, p53, PAP, PAX3,PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras,RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β,TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3. In otherembodiments, the second antigen-binding specificity binds to a tumorantigen that is present on tumor cells specific to, but not limited to,renal cell carcinoma, prostate cancer, colorectal cancer, melanoma,breast cancer, kidney cancer, ovarian cancer, and pancreatic cancer. Theantibodies of the invention, in this aspect, may inhibit the activity ofPD-L1.

In another aspect, the invention provides multi-specific antigen-bindingmolecules or antigen-binding fragments thereof wherein the secondantigen-binding specificity binds to an antigen specific to avirally-infected cell. In certain embodiments, the secondantigen-binding specificity binds to an antigen specific to a cellinfected with a virus selected from the group consisting of HIV, HBV,HCV, HPV, LCMV and SIV.

In another aspect, the invention provides multi-specific antigen-bindingmolecules or antigen-binding fragments thereof comprising a firstantigen-binding specificity that binds to PD-L1 and a secondantigen-binding specificity that binds to a T-cell co-inhibitor such asPD-1, LAG-3, TIM3, B7-1, CTLA-4, BTLA, CD-28, 2B4, LY108, TIGIT, LAIR1,ICOS and CD160.

Any of the multi-specific antigen-binding molecules, or variantsthereof, may be constructed using standard molecular biologicaltechniques (e.g., recombinant DNA and protein expression technology), aswill be well known to a person of ordinary skill in the art.

In some embodiments, PD-L1-specific antibodies are generated in abi-specific format (a “bi-specific”) in which variable regions bindingto distinct domains of PD-L1 are linked together to confer dual-domainspecificity within a single binding molecule. Appropriately designedbi-specifics may enhance overall PD-L1 inhibitory efficacy throughincreasing both specificity and binding avidity. Variable regions withspecificity for individual domains, (e.g., segments of the N-terminaldomain), or that can bind to different regions within one domain, arepaired on a structural scaffold that allows each region to bindsimultaneously to the separate epitopes, or to different regions withinone domain. In one example for a bi-specific, heavy chain variableregions (V_(H)) from a binder with specificity for one domain arerecombined with light chain variable regions (V_(L)) from a series ofbinders with specificity for a second domain to identify non-cognateV_(L) partners that can be paired with an original V_(H) withoutdisrupting the original specificity for that V_(H). In this way, asingle V_(L) segment (e.g., V_(L)1) can be combined with two differentV_(H) domains (e.g., V_(H)1 and V_(H)2) to generate a bi-specificcomprised of two binding “arms” (V_(H)1-V_(L)1 and V_(H)2-V_(L)1). Useof a single V_(L) segment reduces the complexity of the system andthereby simplifies and increases efficiency in cloning, expression, andpurification processes used to generate the bi-specific (See, forexample, U.S. Ser. No. 13/022,759 and US2010/0331527).

Alternatively, antibodies that bind more than one domains and a secondtarget, such as, but not limited to, for example, a second differentanti-PD-L1 antibody, may be prepared in a bi-specific format usingtechniques described herein, or other techniques known to those skilledin the art. Antibody variable regions binding to distinct regions may belinked together with variable regions that bind to relevant sites on,for example, the extracellular domain of PD-L1, to confer dual-antigenspecificity within a single binding molecule. Appropriately designedbi-specifics of this nature serve a dual function. Variable regions withspecificity for the extracellular domain are combined with a variableregion with specificity for outside the extracellular domain and arepaired on a structural scaffold that allows each variable region to bindto the separate antigens.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E,L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221by EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mabe bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-PD-L1 antibodies or antigen-binding fragments thereof of thepresent invention. Therapeutic compositions in accordance with theinvention will be administered with suitable carriers, excipients, andother agents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When an antibody of the present inventionis used for treating a disease or disorder in an adult patient, or forpreventing such a disease, it is advantageous to administer the antibodyof the present invention normally at a single dose of about 0.1 to about100 mg/kg body weight, more preferably about 5 to about 80, about 10 toabout 60, or about 20 to about 50 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. In certain embodiments, the antibody orantigen-binding fragment thereof of the invention can be administered asan initial dose of at least about 0.1 mg to about 800 mg, about 1 toabout 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, toabout 100 mg, or to about 50 mg. In certain embodiments, the initialdose may be followed by administration of a second or a plurality ofsubsequent doses of the antibody or antigen-binding fragment thereof inan amount that can be approximately the same or less than that of theinitial dose, wherein the subsequent doses are separated by at least 1day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; atleast 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; atleast 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks;or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies of the presentinvention is also contemplated herein. Antibody-conjugated nanoparticlesmay be used both for therapeutic and diagnostic applications.Antibody-conjugated nanoparticles and methods of preparation and use aredescribed in detail by Arruebo, M., et al. 2009 (“Antibody-conjugatednanoparticles for biomedical applications” in J. Nanomat. Volume 2009,Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporatedherein by reference. Nanoparticles may be developed and conjugated toantibodies contained in pharmaceutical compositions to target tumorcells or autoimmune tissue cells or virally infected cells.Nanoparticles for drug delivery have also been described in, forexample, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995, eachincorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous, intracranial, intraperitoneal andintramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by methods publicly known. For example, theinjectable preparations may be prepared, e.g., by dissolving, suspendingor emulsifying the antibody or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatesolubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct ofhydrogenated castor oil)], etc. As the oily medium, there are employed,e.g., sesame oil, soybean oil, etc., which may be used in combinationwith a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.The injection thus prepared is preferably filled in an appropriateampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the antibodyis contained in about 5 to about 100 mg and in about 10 to about 250 mgfor the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the present invention are useful for the treatment,prevention, and/or amelioration of disease or disorder or condition suchas cancer, autoimmune disease or a viral infection and/or forameliorating at least one symptom associated with such disease, disorderor condition. In some embodiments of the invention, the antibodiesdescribed herein are useful for treating subjects suffering from primaryor recurrent cancer, including for example, renal cell carcinoma,prostate cancer, ovarian cancer, kidney cancer, colorectal cancer,gastric cancer, breast cancer, head and neck cancer, non-small-cell lungcancer, brain cancer, multiple myeloma, and melanoma. The antibodies maybe used to treat early stage or late-stage symptoms of cancer. In oneembodiment, an antibody or fragment thereof of the invention may be usedto treat metastatic cancer. The antibodies are useful in reducing orinhibiting or shrinking tumor growth of both solid tumors and bloodcancers. In certain embodiments, the antibodies may be used to preventrelapse of a tumor. In certain embodiments, treatment with an antibodyor antigen-binding fragment thereof of the invention may lead to morethan 50% regression, more than 60% regression, more than 70% regression,more than 80% regression or more than 90% regression of a tumor in asubject. In certain embodiments, the antibodies may be used to increasesurvival of a subject suffering from cancer.

In certain embodiments, the antibodies of the invention are useful totreat subjects suffering from a chronic viral infection. In someembodiments, the antibodies of the invention are useful in decreasingviral titers in the host and/or rescuing exhausted T-cells. In oneembodiment, an antibody or antigen-binding fragment thereof theinvention may be administered at a therapeutic dose to a patient with aninfection by human immunodeficiency virus (HIV) or human papilloma virus(HPV) or hepatitis B/C virus (HBV/HCV). In a related embodiment, anantibody or antigen-binding fragment thereof of the invention may beused to treat an infection by simian immunodeficiency virus (SIV) in asimian subject such as cynomolgus. In another embodiment, an antibody orfragment thereof of the invention may be used to treat chronic viralinfection by lymphocytic choriomeningitis virus (LCMV).

In certain embodiments, a blocking antibody of the present invention maybe administered in a therapeutically effective amount to a subjectsuffering from cancer or a viral infection.

In certain embodiments, the antibodies of the invention are useful fortreating an autoimmune disease, including but not limited to, alopeciaareata, autoimmune hepatitis, celiac disease, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia,inflammatory bowel disease, inflammatory myopathies, multiple sclerosis,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma,Sjögren's syndrome, systemic lupus erthyematosus, vitiligo, autoimmunepancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura,Crohn's disease, diabetes type I, eosinophilic fasciitis, eosinophilicenterogastritis, Goodpasture's syndrome, myasthenia gravis, psoriaticarthritis, rheumatic fever, ulcerative colitis, vasculitis and Wegener'sgranulomatosis. In certain embodiments, an activating antibody of theinvention may be used to treat a subject suffering from autoimmunedisease.

One or more antibodies of the present invention may be administered torelieve or prevent or decrease the severity of one or more of thesymptoms or conditions of the disease or disorder.

It is also contemplated herein to use one or more antibodies of thepresent invention prophylactically to patients at risk for developing adisease or disorder such as cancer, and chronic viral infection.

In a further embodiment of the invention the present antibodies are usedfor the preparation of a pharmaceutical composition for treatingpatients suffering from cancer, autoimmune disease or viral infection.In another embodiment of the invention, the present antibodies are usedas adjunct therapy with any other agent or any other therapy known tothose skilled in the art useful for treating cancer, autoimmune diseaseor viral infection.

Combination Therapies

Combination therapies may include an anti-PD-L1 antibody of theinvention and any additional therapeutic agent that may beadvantageously combined with an antibody of the invention, or with abiologically active fragment of an antibody of the invention.

The antibodies of the present invention may be combined synergisticallywith one or more anti-cancer drugs or therapy used to treat cancer,including, for example, renal cell carcinoma, ovarian cancer, prostatecancer, colorectal cancer, non-small-cell lung cancer, and melanoma. Itis contemplated herein to use anti-PD-L1 antibodies of the invention incombination with immunostimulatory and/or immunosupportive therapies toinhibit tumor growth, and/or enhance survival of cancer patients. Theimmunostimulatory therapies include direct immunostimulatory therapiesto augment immune cell activity by either “releasing the brake” onsuppressed immune cells or “stepping on the gas” to activate an immuneresponse. Examples include targeting other checkpoint receptors,vaccination and adjuvants. The immunosupportive modalities may increaseantigenicity of the tumor by promoting immunogenic cell death,inflammation or have other indirect effects that promote an anti-tumorimmune response. Examples include radiation, chemotherapy,anti-angiogenic agents, and surgery.

In various embodiments, one or more antibodies of the present inventionmay be used in combination with a second antibody to PD-L1, an antibodyto PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g.,ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, aCD47 inhibitor, an antagonist of another T-cell co-inhibitor or ligand(e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA),an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelialgrowth factor (VEGF) antagonist [e.g., a “VEGF-Trap” such as afliberceptor other VEGF-inhibiting fusion protein as set forth in U.S. Pat. No.7,087,411, or an anti-VEGF antibody or antigen binding fragment thereof(e.g., bevacizumab, or ranibizumab) or a small molecule kinase inhibitorof VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2inhibitor (e.g., nesvacumab), a transforming growth factor beta (TGFβ)inhibitor, an epidermal growth factor receptor (EGFR) inhibitor (e.g.,erlotinib, cetuximab), an agonist to a co-stimulatory receptor (e.g., anagonist to glucocorticoid-induced TNFR-related protein), an antibody toa tumor-specific antigen (e.g., CA9, CA125, melanoma-associated antigen3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK,prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), a vaccine(e.g., Bacillus Calmette-Guerin, a cancer vaccine), an adjuvant toincrease antigen presentation (e.g., granulocyte-macrophagecolony-stimulating factor), a bispecific antibody (e.g., CD3×CD20bispecific antibody, PSMAxCD3 bispecific antibody), a cytotoxin, achemotherapeutic agent (e.g., dacarbazine, temozolomide,cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin,carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin,paclitaxel, and vincristine), cyclophosphamide, radiotherapy, an IL-6Rinhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g., dupilumab), anIL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21, and IL-15, anantibody-drug conjugate (ADC) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4ADC), an anti-inflammatory drug (e.g., corticosteroids, andnon-steroidal anti-inflammatory drugs), a dietary supplement such asanti-oxidants or any palliative care to treat cancer. In certainembodiments, the anti-PD-L1 antibodies of the present invention may beused in combination with cancer vaccines including dendritic cellvaccines, oncolytic viruses, tumor cell vaccines, etc. to augment theanti-tumor response. Examples of cancer vaccines that can be used incombination with anti-PD-L1 antibodies of the present invention includeMAGE3 vaccine for melanoma and bladder cancer, MUC1 vaccine for breastcancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (includingglioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers). In certainembodiments, the anti-PD-L1 antibodies of the present invention may beused in combination with a dietary supplement such as anti-oxidants orany palliative care to treat cancer.

In certain embodiments, the anti-PD-L1 antibodies of the invention maybe administered in combination with radiation therapy in methods togenerate long-term durable anti-tumor responses and/or enhance survivalof patients with cancer. In some embodiments, the anti-PD-L1 antibodiesof the invention may be administered prior to, concomitantly or afteradministering radiation therapy to a cancer patient. For example,radiation therapy may be administered in one or more doses to tumorlesions followed by administration of one or more doses of anti-PD-L1antibodies of the invention. In some embodiments, radiation therapy maybe administered locally to a tumor lesion to enhance the localimmunogenicity of a patient's tumor (adjuvinating radiation) and/or tokill tumor cells (ablative radiation) followed by systemicadministration of an anti-PD-L1 antibody of the invention. For example,intracranial radiation may be administered to a patient with braincancer (e.g., glioblastoma multiforme) along with systemicadministration of an anti-PD-L1 antibody of the invention. In certainembodiments, the anti-PD-L1 antibodies of the invention may beadministered in combination with radiation therapy and achemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g.,aflibercept).

The antibodies or fragments thereof of the invention may be administeredin combination with one or more anti-viral drugs known in the art,including but not limited to, zidovudine, lamivudine, abacavir,ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine andcorticosteroids. In some embodiments, the anti-PD-L1 antibodies of theinvention may be administered in combination with a LAG3 inhibitor, aCTLA-4 inhibitor, a PD-1 inhibitor or any antagonist of another T-cellco-inhibitor to treat chronic viral infection.

The antibodies of fragments thereof of the invention may be used incombination with any drug or therapy known in the art (e.g.,corticosteroids and other immunosuppressants) to treat an autoimmunedisease or disorder including, but not limited to, alopecia areata,autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hemolytic anemia, inflammatory boweldisease, inflammatory myopathies, multiple sclerosis, primary biliarycirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic lupus erthyematosus, vitiligo, autoimmunepancreatitis, autoimmune urticaira, autoimmune thrombocytopenic purpura,Crohn's disease, diabetes type I, eosinophilic fasciitis, eosinophilicenterogastritis, Goodpasture's syndrome, myasthenia gravis, psoriaticarthritis, rheumatic fever, ulcerative colitis, vasculitis and Wegener'sgranulomatosis.

The additional therapeutically active component(s) may be administeredprior to, concurrent with, or after the administration of the anti-PD-L1antibody of the present invention. For purposes of the presentdisclosure, such administration regimens are considered theadministration of an anti-PD-L1 antibody “in combination with” a secondtherapeutically active component.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-PD-L1 antibody of thepresent invention. For example, a first component may be deemed to beadministered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of an anti-PD-L1 antibody of the present invention. Forexample, a first component may be deemed to be administered “after” asecond component if the first component is administered 1 minute after,5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after,6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hoursafter, 60 hours after, 72 hours after administration of the secondcomponent. In yet other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject concurrent withadministration of an anti-PD-L1 antibody of the present invention.“Concurrent” administration, for purposes of the present invention,includes, e.g., administration of an anti-PD-L1 antibody and anadditional therapeutically active component to a subject in a singledosage form (e.g., co-formulated), or in separate dosage formsadministered to the subject within about 30 minutes or less of eachother. If administered in separate dosage forms, each dosage form may beadministered via the same route (e.g., both the anti-PD-L1 antibody andthe additional therapeutically active component may be administeredintravenously, subcutaneously, etc.); alternatively, each dosage formmay be administered via a different route (e.g., the anti-PD-L1 antibodymay be administered intravenously, and the additional therapeuticallyactive component may be administered subcutaneously). In any event,administering the components in a single dosage from, in separate dosageforms by the same route, or in separate dosage forms by different routesare all considered “concurrent administration,” for purposes of thepresent disclosure. For purposes of the present disclosure,administration of an anti-PD-L1 antibody “prior to”, “concurrent with,”or “after” (as those terms are defined herein above) administration ofan additional therapeutically active component is consideredadministration of an anti-PD-L1 antibody “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which ananti-PD-L1 antibody of the present invention is co-formulated with oneor more of the additional therapeutically active component(s) asdescribed elsewhere herein using a variety of dosage combinations.

In exemplary embodiments in which an anti-PD-L1 antibody of theinvention is administered in combination with a VEGF antagonist (e.g., aVEGF trap such as aflibercept), including administration ofco-formulations comprising an anti-PD-L1 antibody and a VEGF antagonist,the individual components may be administered to a subject and/orco-formulated using a variety of dosage combinations. For example, theanti-PD-L1 antibody may be administered to a subject and/or contained ina co-formulation in an amount selected from the group consisting of 0.01mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg,0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg,3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg,and 10.0 mg; and the VEGF antagonist (e.g., a VEGF trap such asaflibercept) may be administered to the subject and/or contained in aco-formulation in an amount selected from the group consisting of 0.1mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8mg, 2.9 mg and 3.0 mg. The combinations/co-formulations may beadministered to a subject according to any of the administrationregimens disclosed elsewhere herein, including, e.g., twice a week, onceevery week, once every 2 weeks, once every 3 weeks, once every month,once every 2 months, once every 3 months, once every 4 months, onceevery 5 months, once every 6 months, etc.

Administrative Regimens

According to certain embodiments of the present invention, multipledoses of an anti-PD-L1 antibody (or a pharmaceutical compositioncomprising a combination of an anti-PD-L1 antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-PD-L1 antibody ofthe invention. As used herein, “sequentially administering” means thateach dose of anti-PD-L1 antibody is administered to the subject at adifferent point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of an anti-PD-L1 antibody, followed byone or more secondary doses of the anti-PD-L1 antibody, and optionallyfollowed by one or more tertiary doses of the anti-PD-L1 antibody. Theanti-PD-L1 antibody may be administered at a dose of between 0.1 mg/kgto about 100 mg/kg.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-PD-L1 antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-PD-L1 antibody, but generally may differ from one another in termsof frequency of administration.

In certain embodiments, however, the amount of anti-PD-L1 antibodycontained in the initial, secondary and/or tertiary doses varies fromone another (e.g., adjusted up or down as appropriate) during the courseof treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5)doses are administered at the beginning of the treatment regimen as“loading doses” followed by subsequent doses that are administered on aless frequent basis (e.g., “maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1%, 2,2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%,12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%,20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-PD-L1 antibody which is administeredto a patient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PD-L1 antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient at a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of, e.g., once a month (e.g., two, three,four, or more loading doses administered once a month), then themaintenance doses may be administered to the patient once every fiveweeks, once every six weeks, once every seven weeks, once every eightweeks, once every ten weeks, once every twelve weeks, etc.).

Diagnostic Uses of the Antibodies

The anti-PD-L1 antibodies of the present invention may be used to detectand/or measure PD-L1 in a sample, e.g., for diagnostic purposes. Someembodiments contemplate the use of one or more antibodies of the presentinvention in assays to detect a disease or disorder such as cancer,autoimmune disease or chronic viral infection. Exemplary diagnosticassays for PD-L1 may comprise, e.g., contacting a sample, obtained froma patient, with an anti-PD-L1 antibody of the invention, wherein theanti-PD-L1 antibody is labeled with a detectable label or reportermolecule or used as a capture ligand to selectively isolate PD-L1 frompatient samples. Alternatively, an unlabeled anti-PD-L1 antibody can beused in diagnostic applications in combination with a secondary antibodywhich is itself detectably labeled. The detectable label or reportermolecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; afluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,β-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure PD-L1 in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in PD-L1 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of either PD-L1 protein,or fragments thereof, under normal or pathological conditions.Generally, levels of PD-L1 in a particular sample obtained from ahealthy patient (e.g., a patient not afflicted with cancer or anautoimmune disease) will be measured to initially establish a baseline,or standard, level of PD-L1. This baseline level of PD-L1 can then becompared against the levels of PD-L1 measured in samples obtained fromindividuals suspected of having a cancer-related condition, or symptomsassociated with such condition.

The antibodies specific for PD-L1 may contain no additional labels ormoieties, or they may contain an N-terminal or C-terminal label ormoiety. In one embodiment, the label or moiety is biotin. In a bindingassay, the location of a label (if any) may determine the orientation ofthe peptide relative to the surface upon which the peptide is bound. Forexample, if a surface is coated with avidin, a peptide containing anN-terminal biotin will be oriented such that the C-terminal portion ofthe peptide will be distal to the surface.

Aspects of the invention relate to use of the disclosed antibodies asmarkers for predicting prognosis of cancer or an autoimmune disorder inpatients. Antibodies of the present invention may be used in diagnosticassays to evaluate prognosis of cancer in a patient and to predictsurvival.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: Generation of Human Antibodies to PD-L1

Human antibodies to PD-L1 were generated using a fragment of PD-L1 thatranges from about amino acids 19-239 of SEQ ID NO: 351 (GenbankAccession No. NP_054862.1). The immunogen was administered directly,with an adjuvant to stimulate the immune response, to a VELOCIMMUNE®mouse comprising DNA encoding human Immunoglobulin heavy and kappa lightchain variable regions. The antibody immune response was monitored by aPD-L1-specific immunoassay. When a desired immune response was achievedsplenocytes were harvested and fused with mouse myeloma cells topreserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce PD-L1-specific antibodies. Using this technique, and theimmunogen described above, several anti-PD-L1 chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained; exemplary antibodies generated in this manner weredesignated as H2M8306N, H2M8307N, H2M8309N, H2M8310N, H2M8312N,H2M8314N, H2M8316N, H2M8317N, H2M8321N, H2M8323N, H2M8718N, H2M8718N2,and H2M8719N.

Anti-PD-L1 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in U.S.2007/0280945A1, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-PD-L1 antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H1H9323P, H1H9327P, H1H9329P, H1H9336P,H1H9344P2, H1H9345P2, H1H9351P2, H1H9354P2, H1H9364P2, H1H9373P2,H1H9382P2, H1H9387P2, and H1H9396P2.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid andNucleotide Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-PD-L1 antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H2M8306N 2 4 6 8 10 12 1416 H2M8307N 18 20 22 24 26 28 30 32 H2M8309N 34 36 38 40 42 44 46 48H2M8310N 50 52 54 56 58 60 62 64 H2M8312N 66 68 70 72 74 76 78 80H2M8314N 82 84 86 88 90 92 94 96 H2M8316N 98 100 102 104 106 108 110 112H2M8317N 114 116 118 120 122 124 126 128 H2M8321N 130 132 134 136 138140 142 144 H2M8323N 146 148 150 152 154 156 158 160 H2M8718N 162 164166 168 170 172 174 176 H2M8718N2 178 180 182 184 170 172 174 176H2M8719N 186 188 190 192 194 196 198 200 H1H9323P 202 204 206 208 210212 214 216 H1H9327P 218 220 222 224 226 228 230 232 H1H9329P 234 236238 240 242 244 246 248 H1H9336P 250 252 254 256 258 260 262 264H1H9344P2 266 268 270 272 274 276 278 280 H1H9345P2 282 284 286 288 274276 278 280 H1H9351P2 290 292 294 296 274 276 278 280 H1H9354P2 298 300302 304 274 276 278 280 H1H9364P2 306 308 310 312 274 276 278 280H1H9373P2 314 316 318 320 274 276 278 280 H1H9382P2 322 324 326 328 274276 278 280 H1H9387P2 330 332 334 336 274 276 278 280 H1H9396P2 338 340342 344 274 276 278 280

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H2M8306N 1 3 57 9 11 13 15 H2M8307N 17 19 21 23 25 27 29 31 H2M8309N 33 35 37 39 41 4345 47 H2M8310N 49 51 53 55 57 59 61 63 H2M8312N 65 67 69 71 73 75 77 79H2M8314N 81 83 85 87 89 91 93 95 H2M8316N 97 99 101 103 105 107 109 111H2M8317N 113 115 117 119 121 123 125 127 H2M8321N 129 131 133 135 137139 141 143 H2M8323N 145 147 149 151 153 155 157 159 H2M8718N 161 163165 167 169 171 173 175 H2M8718N2 177 179 181 183 169 171 173 175H2M8719N 185 187 189 191 193 195 197 199 H1H9323P 201 203 205 207 209211 213 215 H1H9327P 217 219 221 223 225 227 229 231 H1H9329P 233 235237 239 241 243 245 247 H1H9336P 249 251 253 255 257 259 261 263H1H9344P2 265 267 269 271 273 275 277 279 H1H9345P2 281 283 285 287 273275 277 279 H1H9351P2 289 291 293 295 273 275 277 279 H1H9354P2 297 299301 303 273 275 277 279 H1H9364P2 305 307 309 311 273 275 277 279H1H9373P2 313 315 317 319 273 275 277 279 H1H9382P2 321 323 325 327 273275 277 279 H1H9387P2 329 331 333 335 273 275 277 279 H1H9396P2 337 339341 343 273 275 277 279

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “H2M,” “H2aM,” etc.), followed by anumerical identifier (e.g. “8306,” “9323,” etc., as shown in Table 1),followed by a “P,” “N,” “P2,” or “N2” suffix. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H2M8306N,” “H1H9344P2,” etc. The H1H, H2M and H2aM prefixes on theantibody designations used herein indicate the particular Fc regionisotype of the antibody. For example, an “H1H” antibody has a human IgG1Fc, an “HIM” antibody has a mouse IgG1 Fc, and an “H2M” or “H2aM”antibody has a mouse IgG2 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 1—will remain the same, and the bindingproperties to antigen are expected to be identical or substantiallysimilar regardless of the nature of the Fc domain.

Example 3: Antibody Binding to PD-L1 as Determined by Surface PlasmonResonance

Binding association and dissociation rate constants (k_(a) and k_(d),respectively), equilibrium dissociation constants and dissociationhalf-lives (K_(D) and t_(1/2), respectively) of antigen binding topurified anti-PD-L1 monoclonal antibodies were determined using areal-time surface plasmon resonance biosensor assay on a Biacore 4000instrument. The Biacore sensor surface was either derivatized withpolyclonal rabbit anti-mouse antibody (GE, #BR-1008-38) or withmonoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to captureapproximately 200-300 RUs of anti-PD-L1 monoclonal antibodies, expressedwith either a mouse Fc or with human Fc, respectively. The PD-L1reagents tested for binding to the anti-PD-L1 antibodies includedrecombinant human PD-L1 (amino acids 19-239 of accession numberNP_054862.1) expressed with a C-terminal myc-myc-hexahistidine tag(hPD-L1-MMH; SEQ ID: 345), recombinant cynomolgus monkey PD-L1 expressedwith a C-terminal myc-myc-hexahistidine tag (MfPD-L1-MMH; SEQ ID: 346),recombinant human PD-L1 (amino acids 19-239 of accession numberNP_054862.1) expressed with either a C-terminal human IgG1 Fc tag(hPD-L1-hFc; SEQ ID: 347) or with a C-terminal mouse IgG2a Fc tag(hPD-L1-mFc; SEQ ID: 348), and recombinant cynomolgus monkey PD-L1expressed with a C-terminal mouse IgG2a Fc tag (MfPD-L1-mFc; SEQ ID:353). Different concentrations of PD-L1 reagents were injected over theanti-PD-L1 monoclonal antibody captured surface at a flow rate of 30μL/min. The binding of the PD-L1 reagents to the captured monoclonalantibodies was monitored for 3 to 4 minutes while the dissociation ofantibody bound PD-L1 reagents was monitored for 10 minutes in HBSTrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/vSurfactant tween-20). Experiments were performed at either 25° C. or 37°C. Kinetic association (k_(a)) and dissociation (k_(d)) rate constantswere determined by processing and fitting the data to a 1:1 bindingmodel using Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werethen calculated from the kinetic rate constants as: K_(D)(M)=k_(d)/k_(a) and t_(1/2) (min)=[In2/(60*k_(d))]. Binding kineticsparameters for different anti-PD-L1 monoclonal antibodies binding todifferent PD-L1 reagents at 25° C. and 37° C. are tabulated in Tables3-8.

TABLE 3 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to hPD-L1-MMH at 25° C. hPD-L1-MMH Monomer Binding at 25° C.k_(a) k_(d) K_(D) t1/2 Antibody (1/Ms) (1/s) (M) (min) H2aM8306N1.44E+05 1.00E−02 6.96E−08 1.2 H2aM8307N 3.27E+04 1.47E−04 4.50E−09 79H2aM8309N 6.42E+05 1.04E−02 1.63E−08 1.1 H2aM8310N 8.86E+04 3.10E−043.50E−09 37 H2aM8312N 7.59E+04 7.22E−04 9.51E−09 16 H2aM8314N 9.25E+055.17E−05 5.58E−11 224 H2aM8316N 9.57E+05 1.12E−03 1.17E−09 10 H2aM8317N9.40E+04 3.95E−02 4.21E−07 0.3 H2aM8321N 1.03E+05 3.59E−03 3.49E−08 3.2H2aM8323N 9.37E+05 2.23E−04 2.38E−10 52 H2aM8718N 9.27E+05 8.01E−058.63E−11 144 H2aM8719N 8.64E+04 2.26E−03 2.62E−08 5.1 H1H9323P 5.51E+041.88E−02 3.41E−07 0.6 H1H9327P 4.19E+05 2.59E−04 6.19E−10 45 H1H9329P1.77E+06 1.34E−01 7.58E−08 0.1 H1H9336P 7.92E+05 3.90E−04 4.92E−10 30H1H9345P2 9.02E+04 1.49E−02 1.65E−07 0.8 H1H9351P2 4.56E+05 8.96E−041.96E−09 13 H1H9354P2 4.76E+04 3.83E−04 8.04E−09 30 H1H9364P2 9.32E+052.99E−04 3.21E−10 39 H1H9373P2 2.65E+06 2.91E−04 1.10E−10 40 H1H9382P27.90E+04 4.79E−03 6.06E−08 2.4 H1H9387P2 2.64E+06 5.82E−02 2.21E−08 0.2H1H9396P2 1.72E+05 2.18E−03 1.27E−08 5.3

TABLE 4 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to hPD-L1-MMH at 37° C. hPD-L1-MMH Monomer Binding at 37° C.k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H2aM8306N1.80E+05 3.10E−02 1.72E−07 0.4 H2aM8307N 5.35E+04 8.79E−04 1.64E−08 13H2aM8309N 1.06E+06 3.14E−02 2.97E−08 0.4 H2aM8310N 1.32E+05 1.28E−039.70E−09 9.0 H2aM8312N 8.89E+04 4.03E−03 4.53E−08 2.9 H2aM8314N 1.07E+061.50E−04 1.40E−10 77 H2aM8316N 1.01E+06 5.30E−03 5.24E−09 2.2 H2aM8317N9.03E+04 5.85E−02 6.47E−07 0.2 H2aM8321N 1.01E+05 9.29E−03 9.16E−08 1.2H2aM8323N 1.38E+06 6.84E−04 4.97E−10 17 H2aM8718N 1.08E+06 1.55E−041.44E−10 74 H2aM8719N 1.50E+05 5.76E−03 3.84E−08 2.0 H1H9323P 1.21E+054.25E−02 3.52E−07 0.3 H1H9327P 5.21E+05 4.29E−04 8.24E−10 27 H1H9329P2.82E+06 3.29E−01 1.17E−07 0.04 H1H9336P 1.07E+06 7.88E−04 7.33E−10 15H1H9345P2 1.72E+05 3.40E−02 1.97E−07 0.3 H1H9351P2 6.82E+05 1.68E−032.47E−09 6.9 H1H9354P2 7.39E+04 1.14E−03 1.54E−08 10 H1H9364P2 1.35E+065.63E−04 4.17E−10 21 H1H9373P2 3.09E+06 5.58E−04 1.80E−10 21 H1H9382P29.97E+04 1.07E−02 1.07E−07 1.1 H1H9387P2 3.49E+06 1.37E−01 3.91E−08 0.08H1H9396P2 3.44E+05 1.13E−02 3.30E−08 1.0

TABLE 5 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to human PD-L1 Dimer at 25° C. Human PD-L1 Dimer Binding at 25°C. k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H2aM8306N7.39E+05 3.09E−04 4.18E−10 37 H2aM8307N 1.89E+04 2.89E−05 1.54E−09 399H2aM8309N 3.36E+06 1.44E−04 4.29E−11 80 H2aM8310N 3.13E+05 5.71E−051.83E−10 202 H2aM8312N 2.47E+05 1.02E−04 4.13E−10 113 H2aM8314N 3.16E+061.35E−05 4.26E−12 859 H2aM8316N 3.08E+06 1.44E−04 4.68E−11 80 H2aM8317N3.59E+05 4.50E−04 1.25E−09 26 H2aM8321N 8.13E+05 2.87E−04 3.53E−10 40H2aM8323N 2.91E+06 2.05E−05 7.04E−12 565 H2aM8718N 3.20E+06 1.62E−055.06E−12 713 H2aM8719N 3.42E+05 2.62E−04 7.67E−10 44 H1H9323P 2.24E+051.69E−04 7.54E−10 68 H1H9327P 4.66E+05 7.87E−05 1.69E−10 147 H1H9329P2.97E+06 7.68E−04 2.59E−10 15 H1H9336P 1.38E+06 1.09E−04 7.86E−11 106H1H9345P2 5.00E+05 2.37E−04 4.74E−10 49 H1H9351P2 9.16E+05 1.53E−041.67E−10 76 H1H9354P2 1.68E+05 1.16E−04 6.89E−10 100 H1H9364P2 2.42E+061.06E−04 4.37E−11 109 H1H9373P2 4.08E+06 1.06E−04 2.60E−11 109 H1H9382P22.23E+05 1.48E−04 6.63E−10 78 H1H9387P2 5.07E+06 2.17E−04 4.27E−11 53H1H9396P2 7.76E+05 1.90E−04 2.45E−10 61

TABLE 6 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to human PD-L1 Dimer at 37° C. Human PD-L1 Dimer Binding at 37°C. k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H2aM8306N9.97E+05 4.16E−04 4.17E−10 28 H2aM8307N 4.41E+04 2.21E−04 5.00E−09 52H2aM8309N 4.41E+06 1.66E−04 3.76E−11 70 H2aM8310N 4.81E+05 1.24E−042.58E−10 93 H2aM8312N 3.57E+05 2.61E−04 7.32E−10 44 H2aM8314N 3.89E+062.30E−05 5.91E−12 503 H2aM8316N 4.06E+06 2.37E−04 5.85E−11 49 H2aM8317N4.81E+05 6.57E−04 1.36E−09 18 H2aM8321N 9.46E+05 2.69E−04 2.85E−10 43H2aM8323N 4.32E+06 1.21E−04 2.80E−11 96 H2aM8718N 3.72E+06 1.98E−055.32E−12 584 H2aM8719N 4.37E+05 2.91E−04 6.66E−10 40 H1H9323P 5.19E+052.03E−04 3.91E−10 57 H1H9327P 6.83E+05 1.36E−04 2.00E−10 85 H1H9329P3.87E+06 2.67E−03 6.89E−10 4.3 H1H9336P 2.75E+06 8.31E−05 3.02E−11 139H1H9345P2 6.82E+05 2.03E−04 2.97E−10 57 H1H9351P2 1.25E+06 1.46E−041.17E−10 79 H1H9354P2 4.56E+05 1.45E−04 3.17E−10 80 H1H9364P2 3.34E+066.96E−05 2.08E−11 166 H1H9373P2 5.12E+06 8.97E−05 1.75E−11 129 H1H9382P24.92E+05 1.37E−04 2.78E−10 84 H1H9387P2 6.12E+06 3.92E−04 6.39E−11 29H1H9396P2 1.09E+06 2.58E−04 2.37E−10 45

TABLE 7 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to MfPD-L1-MMH at 25° C. MfPD-L1-MMH Monomer Binding at 25° C.k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H2aM8306N1.65E+05 7.88E−03 4.77E−08 1.5 H2aM8307N 2.61E+04 1.07E−03 4.09E−08 11H2aM8309N 8.70E+05 1.30E−02 1.49E−08 0.9 H2aM8310N 1.03E+05 3.06E−042.97E−09 38 H2aM8312N 7.09E+04 5.97E−04 8.42E−09 19 H2aM8314N 9.66E+057.00E−05 7.24E−11 165 H2aM8316N 9.71E+05 1.64E−03 1.69E−09 7.0 H2aM8317N1.06E+05 2.38E−02 2.26E−07 0.5 H2aM8321N 1.34E+05 4.02E−03 2.99E−08 2.9H2aM8323N 5.47E+05 8.68E−03 1.59E−08 1.3 H2aM8718N 9.04E+05 6.64E−057.35E−11 174 H2aM8719N 8.17E+04 2.68E−03 3.28E−08 4.3 H1H9323P 8.22E+042.40E−02 2.92E−07 0.5 H1H9327P 3.59E+05 3.33E−04 9.28E−10 35 H1H9329P1.76E+06 1.35E−01 7.69E−08 0.09 H1H9336P 6.79E+05 5.94E−04 8.76E−10 19H1H9345P2 1.10E+05 8.50E−03 7.73E−08 1.4 H1H9351P2 3.49E+05 1.11E−033.19E−09 10 H1H9354P2 4.60E+04 3.05E−04 6.64E−09 38 H1H9364P2 7.57E+053.12E−04 4.12E−10 37 H1H9373P2 2.21E+06 2.82E−04 1.27E−10 41 H1H9382P28.22E+04 1.29E−02 1.57E−07 0.9 H1H9387P2 2.37E+06 6.04E−02 2.55E−08 0.2H1H9396P2 2.06E+05 2.52E−03 1.22E−08 4.6

TABLE 8 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to MfPD-L1-MMH at 37° C. MfPD-L1-MMH Monomer Binding at 37° C.k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H2aM8306N2.74E+05 3.68E−02 1.34E−07 0.3 H2aM8307N 1.87E+05 1.43E−03 7.63E−09 8.1H2aM8309N 1.07E+06 2.81E−02 2.63E−08 0.4 H2aM8310N 4.71E+05 1.07E−032.27E−09 11 H2aM8312N 1.01E+05 3.32E−03 3.27E−08 3.5 H2aM8314N 1.07E+061.52E−04 1.42E−10 76 H2aM8316N 1.02E+06 5.38E−03 5.27E−09 2.1 H2aM8317N2.66E+05 4.76E−02 1.79E−07 0.2 H2aM8321N 1.59E+05 8.16E−03 5.11E−08 1.4H2aM8323N 9.56E+05 2.82E−02 2.95E−08 0.4 H2aM8718N 1.10E+06 1.46E−041.33E−10 79 H2aM8719N 1.35E+05 6.99E−03 5.19E−08 1.7 H1H9323P 1.25E+054.99E−02 3.98E−07 0.2 H1H9327P 4.77E+05 5.34E−04 1.12E−09 22 H1H9329P2.66E+06 3.64E−01 1.37E−07 0.03 H1H9336P 9.09E+05 1.25E−03 1.38E−09 9.2H1H9345P2 1.64E+05 1.97E−02 1.21E−07 0.6 H1H9351P2 5.60E+05 2.01E−033.59E−09 5.8 H1H9354P2 8.59E+04 8.44E−04 9.82E−09 14 H1H9364P2 1.12E+066.33E−04 5.66E−10 18 H1H9373P2 2.81E+06 5.69E−04 2.03E−10 20 H1H9382P21.30E+05 2.45E−02 1.89E−07 0.5 H1H9387P2 3.20E+06 1.57E−01 4.89E−08 0.07H1H9396P2 3.94E+05 1.20E−02 3.05E−08 1.0

TABLE 9 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to monkey PD-L1-mFc at 25° C. MfPD-L1-mFc Binding at 25° C.k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H1H8314N2.58E+06 6.77E−05 2.63E−11 171 H1H8316N 2.71E+06 5.23E−05 1.93E−11 221H1H8323N 2.67E+06 6.16E−05 2.31E−11 188 H1H9351P2 7.81E+05 7.19E−059.22E−11 161 H1H9364P2 1.32E+06 7.75E−05 5.87E−11 149 H1H9373P2 2.85E+064.96E−05 1.74E−11 233 H1H9387P2 3.55E+06 1.61E−04 4.52E−11 72 H1H9351P28.64E+05 7.80E−05 9.03E−11 148 H1H9364P2 1.25E+06 5.80E−05 4.62E−11 199H1H9373P2 3.27E+06 6.55E−05 2.00E−11 176 H1H9387P2 2.90E+06 2.12E−047.30E−11 55 H1H9323P 2.80E+05 1.45E−04 5.19E−10 79 H1H9327P 3.48E+051.02E−04 2.94E−10 113 H1H9329P 1.74E+06 6.72E−04 3.86E−10 17 H1H9336P9.90E+05 6.16E−05 6.22E−11 188 H1H9344P2 3.50E+05 1.38E−04 3.93E−10 84H1H9345P2 2.96E+05 1.51E−04 5.12E−10 76 H1H9354P2 2.05E+05 9.14E−054.47E−10 126 H1H9382P2 2.13E+05 1.33E−04 6.26E−10 87 H1H9396P2 8.37E+051.88E−04 2.25E−10 61

TABLE 10 Binding Kinetics parameters of anti-PD-L1 monoclonal antibodiesbinding to monkey PD-L1-mFc at 37° C. MfPD-L1-mFc Binding at 37° C.k_(a) k_(d) K_(D) t_(1/2) Antibody (1/Ms) (1/s) (M) (min) H1H8314N2.89E+06 9.60E−05 3.33E−11 120 H1H8316N 2.96E+06 6.16E−05 2.08E−11 187H1H8323N 2.99E+06 1.35E−04 4.51E−11 86 H1H9351P2 1.06E+06 9.46E−058.91E−11 122 H1H9364P2 2.36E+06 1.11E−04 4.71E−11 104 H1H9373P2 3.15E+068.59E−05 2.73E−11 134 H1H9387P2 3.41E+06 4.74E−04 1.39E−10 24 H1H9351P21.61E+06 1.04E−04 6.47E−11 111 H1H9364P2 2.41E+06 6.76E−05 2.80E−11 171H1H9373P2 3.86E+06 1.23E−04 3.19E−11 94 H1H9387P2 2.90E+06 4.65E−041.61E−10 25 H1H9323P 3.84E+05 2.44E−04 6.36E−10 47 H1H9327P 7.64E+052.94E−04 3.85E−10 39 H1H9329P 2.18E+06 1.54E−03 7.08E−10 8 H1H9336P1.86E+06 4.60E−05 2.47E−11 251 H1H9344P2 9.05E+05 2.17E−04 2.40E−10 53H1H9345P2 8.61E+05 2.92E−04 3.39E−10 40 H1H9354P2 2.72E+05 2.03E−047.46E−10 57 H1H9382P2 2.84E+05 2.35E−04 8.25E−10 49 H1H9396P2 1.57E+065.02E−04 3.19E−10 23

As shown in Table 3, at 25° C., all 25 anti-PD-L1 antibodies of theinvention bound to hPD-L1-MMH with K_(D) values ranging from 55.8 pM to421 nM. As shown in Table 4, at 37° C., all 25 anti-PD-L1 antibodies ofthe invention bound to hPD-L1-MMH with K_(D) values ranging from 140 pMto 647 nM. As shown in Table 5, at 25° C., all 25 anti-PD-L1 antibodiesof the invention bound to hPD-L1 dimer with K_(D) values ranging from4.26 pM to 1.54 nM. As shown in Table 6, at 37° C., all 25 anti-PD-L1antibodies of the invention bound to hPD-L1 dimer with K_(D) valuesranging from 5.32 pM to 5.0 nM. As shown in Table 7, at 25° C., all 25anti-PD-L1 antibodies of the invention bound to MfPD-L1-MMH with K_(D)values ranging from 72.4 pM to 292 nM. As shown in Table 8, at 37° C.,all 25 anti-PD-L1 antibodies of the invention bound to MfPD-L1-MMH withK_(D) values ranging from 133 pM to 398 nM. As shown in Table 9, at 25°C., all 20 anti-PD-L1 antibodies of the invention tested bound toMfPD-L1-mFc with K_(D) values ranging from 17.4 pM to 626 pM. As shownin Table 10, at 37° C., all 20 anti-PD-L1 antibodies of the inventiontested bound to MfPD-L1-mFc with K_(D) values ranging from 20.8 pM to825 pM.

Example 4: Blocking of PD-L1 Binding to PD-1 as Determined by ELISA

The ability of monoclonal anti-PD-L1 antibodies to block human PD-L1from binding to its binding partners, the human PD-1 and the human B7-1receptors, was measured using two competition sandwich ELISA formats.

A dimeric human PD-1 protein comprised of a portion of the extracellulardomain (amino acids 25-170 of accession number NP_005009.2 with a C93Schange) that was expressed with a C-terminal hFc tag (hPD-1-hFc; SEQ ID:350) and a dimeric human B7-1 protein comprised of a portion of theextracellular domain expressed with C-terminal hFc tag and hexahistidinetags (hB7-1-hFc-6His; R&D Systems, #140-B1) were separately coated at 2μg/mL on a 96-well microtiter plate in a PBS buffer overnight at 4° C.Nonspecific binding sites were subsequently blocked using a 0.5% (w/v)solution of BSA in PBS. A constant amount of 0.5 nM or 8.0 nM of adimeric hPD-L1 protein comprised of a portion of the human PD-L1extracellular domain that was expressed with a C-terminal mFc tag(hPD-L1-mFc; SEQ ID: 348) was separately titrated with concentrations ofanti-PD-1 antibodies and isotype control antibodies ranging between0-210 nM in serial dilution. These antibody-protein complexes were thenincubated for 1 hour at room temperature (RT). Complexes with 0.5 nMconstant hPD-L1-mFc were subsequently transferred to microtiter platescoated with hPD-1-hFc, and complexes with 8 nM constant hPD-L1-mFc weretransferred to hB7-1-hFc-6His coated plates. The complexes were allowedto bind to the coated plates for 1 hour at RT. After the 1 hourincubation, the wells were washed and plate-bound hPD-L1-mFc wasdetected with an anti-mFc polyclonal antibody conjugated withhorse-radish peroxidase (Jackson ImmunoResearch, #115-035-164). Sampleswere developed with a TMB solution (BD Biosciences, #51-2606KC and#51-2607KC) to produce a colorimetric reaction and neutralized with 1Msulfuric acid before measuring absorbance at 450 nm on a Victor X5 platereader. Data analysis used a sigmoidal dose-response model within Prism™software. The calculated IC₅₀ value, defined as the concentration ofantibody required to block 50% of hPD-L1-mFc binding to hPD-1-hFc orhB7-1-hFc-6His, was used as an indicator of blocking potency. Maximumblocking values represent the ability of the antibodies to blockhPD-L1-mFc binding relative to baseline. The absorbance measured at theconstant amount of hPD-L1 on the dose curve is defined as 0% blockingand the absorbance with no added hPD-L1 is defined as 100% blocking. Theabsorbance values of the wells containing the highest concentration foreach antibody were used to determine the blocking percent at maximumconcentration antibody tested. Antibodies with a maximum percentblockade below 25% were characterized as non-blockers, and their IC₅₀values were not reported in Table 11. Antibodies with a maximum percentblockade below −25% were characterized as non-blockers/enhancers.

TABLE 11 ELISA blocking of hPD-L1-mFc binding to hPD-1-hFc andhB7-1-hFc-6His by anti- PD-L1 antibodies Blocking 8 nM of Blocking 0.5Highest hPD-L1- Highest Blocking 0.5 nM of Antibody Blocking 8 nM mFcAntibody nM of hPD-L1-mFc concentration of binding to concentrationhPD-L1-mFc binding to (nM) tested hPD-L1-mFc hB7-1-hFc- (nM) testedbinding to hPD-1-hFc, on hB7-1- binding to 6His, % on hPD-1- hPD-1-hFc,% maximum hFc-6His hB7-1-hFc- maximum Antibody hFc coat IC₅₀ (M)blocking coat 6His, IC₅₀ (M) blocking H2aM8306N 50 <2.5E−10 (*) 99 150<4.0E−09 (*) 93 H2aM8307N 50 IC 29 150 NBI/enhancer −44 H2aM8309N 50<2.5E−10 (*) 98 150 <4.0E−09 (*) 98 H2aM8310N 50 3.9E−10  76 150<4.0E−09 (*) 98 H2aM8312N 50 NBI 23 150 <4.0E−09(*) 98 H2aM8314N 50<2.5E−10 (*) 93 150 <4.0E−09 (*) 97 H2aM8316N 50 <2.5E−10 (*) 96 150<4.0E−09 (*) 98 H2aM8317N 50 NBI −11 150 NBI/enhancer −166 H2aM8321N 50NBI 10 150 NBI/enhancer −124 H2aM8323N 50 <2.5E−10 (*) 100 150 <4.0E−09(*) 99 H2aM8718N 50 <2.5E−10 (*) 97 150 1.0E−08  57 H2aM8719N 507.7E−10  95 150 NBI/enhancer −50 H1H9323P 50 <2.5E−10 (*) 43 210NBI/enhancer −25 H1H9327P 50 NBI/enhancer −28 210 IC 41 H1H9329P 50<2.5E−10 (*) 100 210 4.9E−09  100 H1H9336P 50 <2.5E−10 (*) 100 2104.4E−09  99 H1H9344P2 50 NBI 15 210 NBI/enhancer −51 H1H9345P2 50<2.5E−10 (*) 26 210 NBI/enhancer −34 H1H9351P2 50 <2.5E−10 (*) 100 2104.4E−09  100 H1H9354P2 50 5.3E−10  34 210 NBI −13 H1H9364P2 50 <2.5E−10(*) 100 210 4.1E−09  101 H1H9373P2 50 <2.5E−10 (*) 100 210 4.6E−09  101H1H9382P2 50 <2.5E−10 (*) 39 210 NBI/enhancer −30 H1H9387P2 50 <2.5E−10(*) 100 210 <4.0E−09 (*) 100 H1H9396P2 50 <2.5E−10 (*) 59 210 NBI 8Isotype 50 NBI −11 210 NBI −3 control- human IgG1 Isotype 50 NBI 5 210NBI −3 control- mouse IgG2a (*) below theoretical bottom of the assay;Assay theoretical bottom is 2.5E−10 M for hPD-1-hFc coat and 4.0E−09 Mfor hB7-1-hFc-6His coat; NBI—non-blocker; IC—inconclusive

As shown in Table 11, 19 of the 25 anti-PD-L1 antibodies of theinvention blocked 0.5 nM of hPD-L1-mFc from binding to hPD-1-hFc withIC₅₀ values ranging from less than 250 pM to 770 pM with maximum percentblockade ranging from 26% to 100%. Four of the 25 anti-PD-L1 antibodiestested were characterized as non-blockers of hPD-L1-mFc binding tohPD-1-hFc, while one antibody tested (H1H9327P) was characterized as anon-blocker/enhancer of hPD-L1-mFc binding to hPD-1-hFc. One antibody(H2aM8307N) demonstrated weak blocking of hPD-L1-mFc binding tohPD-1-hFc with a maximum percent blockade of 29%; however the IC₅₀ valuecould not be determined for this sample.

Further, 14 of the 25 anti-PD-L1 antibodies of the invention blocked 8nM of hPD-L1-mFc from binding to hB7-1-hFc-6His with IC₅₀ values rangingfrom <4 nM to 10 nM with maximum percent blockade ranging from 57% to101%. Two of the 25 anti-PD-L1 antibodies tested were characterized asnon-blockers of hPD-L1-mFc binding to hB7-1-hFc-6His, while 8 antibodiestested were characterized as non-blockers/enhancers of hPD-L1-mFcbinding to hB7-1-hFc-6His. One antibody (H1H9327P) demonstrated weakblocking of hPD-L1-mFc binding to hB7-1-hFc-6His with a maximum percentblockade of 41%; however the IC₅₀ value could not be determined for thissample.

Example 5: Blocking of PD-L1 Binding to PD-1 as Determined by BiosensorAssay and by Surface Plasmon Resonance

Inhibition of human PD-L1 from binding to human PD-1 by differentanti-PD-L1 monoclonal antibodies was studied either using real timebio-layer interferometry assay on an Octet Red96 biosensor instrument(Fortebio Inc.) or using a real-time surface plasmon resonance biosensorassay on a Biacore 3000 instrument.

Inhibition studies for anti-PD-L1 monoclonal antibodies expressed withmouse Fc were performed on an Octet Red96 instrument. First, 100 nM of arecombinant human PD-L1 expressed with a C-terminal mouse IgG2a Fc tag(hPD-L1-mFc; SEQ ID: 348) was incubated with 500 nM of each anti-PD-L1monoclonal antibody for at least 1 hour before running the inhibitionassay. Approximately 0.8 nm to 1.2 nm of recombinant human PD-1expressed with a C-terminal human IgG1 Fc tag (hPD-1-hFc; SEQ ID: 350)was captured using anti-human IgG Fc capture biosensors. The Octetbiosensors captured with hPD-1-hFc were subsequently submerged intowells containing the mixture of hPD-L1-mFc and different anti-PD-L1monoclonal antibodies. The entire experiment was performed at 25° C. inOctet HBST buffer (0.01 M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/vSurfactant P20, 0.1 mg/mL BSA) with a plate shaking at a speed of 1000rpm. The biosensors were washed in Octet HBST buffer in between eachstep of the experiment. The real-time binding response was monitoredduring the course of the experiment and the binding response at the endof every step was recorded. Binding of hPD-L1-mFc to the capturedhPD-1-hFc was compared in the presence and absence of differentanti-PD-L1 monoclonal antibodies and was used to determine the blockingbehavior of the tested antibodies as shown in Table 12.

TABLE 12 Inhibition of human PD-1 binding to human PD-L1 by differentanti-PD-L1 monoclonal antibodies expressed with mouse Fc performed onOctet Red96 instrument. Binding of the mixture of Anti-PD-L1 Amount of100 nM hPD-L1-mFc and monoclonal hPD-1-hFc 500 nM anti-PD-L1 % antibodyCaptured (nm) monoclonal antibody (nm) Blocking No antibody 1.05 0.31 0H2aM8306N 0.98 0.02 94 H2aM8307N 1.01 0.46 −48 H2aM8309N 0.89 0.02 94H2aM8310N 0.95 0.13 58 H2aM8312N 1.06 0.52 −68 H2aM8314N 0.99 0.02 94H2aM8316N 1.06 0.01 97 H2aM8317N 0.92 0.58 −87 H2aM8321N 1.04 0.60 −94H2aM8323N 1.00 0.02 94 H2aM8718N 1.08 0.01 97 H2aM8719N 0.93 0.11 65Isotype control antibody 1.10 0.35 −13

As shown in Table 12, 8 of the 12 anti-PD-L1 antibodies tested on theOctet Red96 instrument demonstrated blocking of hPD-L1-mFc from bindingto hPD-1-hFc ranging from 58% to 97%. Four anti-PD-L1 antibodies testeddemonstrated the ability to enhance the binding of hPD-L1-mFc tohPD-1-hFc.

Next, inhibition studies for anti-PD-L1 monoclonal antibodies expressedwith human Fc were performed on Biacore 3000 instrument. First, 100 nMof recombinant human PD-L1 expressed with a C-terminal human IgG1 Fc tag(hPD-L1-hFc; SEQ ID: 350) was incubated with 500 nM of each anti-PD-L1monoclonal antibody for at least 1 hour before running the inhibitionassay. A CM5 Biacore sensor surface was first derivatized withanti-mouse IgG2a specific polyclonal antibody (Southern Biotech,#1080-01) using the standard EDC-NHS chemistry. Around 230 RUs ofrecombinant human PD-1 expressed with a C-terminal mouse IgG2a Fc tag(hPD-1-mFc; SEQ ID: 348) was then captured and was followed by aninjection of 100 nM of hPD-L1-hFc in the presence and absence ofdifferent anti-PD-L1 monoclonal antibodies at a flow rate of 25 μL/minfor 2 minutes. The entire experiment was performed at 25° C. in HBSTrunning buffer (0.01 M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/vSurfactant P20). The real-time binding responses were monitored duringthe entire course of the experiment and the binding response at the endof every step was recorded. Binding of hPD-L1-hFc to the capturedhPD-1-mFc was compared in the presence and absence of differentanti-PD-L1 monoclonal antibodies and was used to determine the blockingbehavior of the tested antibodies as shown in Table 13.

TABLE 13 Inhibition of human PD-1 binding to human PD-L1 by differentanti-PD-L1 monoclonal antibodies expressed with human Fc performed onBiacore 3000 instrument. Binding of the mixture of Anti-PD-L1 Amount of100 nM hPD-L1-hFc and monoclonal hPD-1-mFc 500 nM anti-PD-L1 % antibodyCaptured (RU) monoclonal antibody (nm) Blocking No mAb 222 56 0 H1H9323P224 262 −368 H1H9327P 226 172 −207 H1H9329P 227 2 97 H1H9336P 227 9 84H1H9345P2 229 292 −422 H1H9351P2 227 6 90 H1H9354P2 229 296 −428H1H9364P2 228 8 86 H1H9373P2 227 6 89 H1H9382P2 228 307 −448 H1H9387P2228 5 91 H1H9396P2 228 164 −193 Isotype control antibody 228 56 0

As shown in Table 13, 6 out of 12 anti-PD-L1 antibodies of the inventiontested on the Biacore 3000 instrument demonstrated blocking ofhPD-L1-hFc from binding to hPD-1-mFc ranging from 84% to 97%. Sixanti-PD-L1 antibodies tested demonstrated the ability to enhance thebinding of hPD-L1-hFc to hPD-1-mFc.

Example 6: Octet Cross-Competition Between Anti-PD-L1 Antibodies

Binding competition between anti-PD-L1 monoclonal antibodies wasdetermined using a real time, label-free bio-layer interferometry assayon an Octet RED384 biosensor (Pall ForteBio Corp.). The entireexperiment was performed at 25° C. in Octet HBST buffer (0.01 M HEPESpH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, 0.1 mg/mL BSA)with the plate shaking at the speed of 1000 rpm. To assess whether 2antibodies were able to compete with one another for binding to theirrespective epitopes on the recombinant human PD-L1 expressed with aC-terminal myc-myc-hexahistidine tag (hPD-L1-MMH; SEQ ID: 345), around˜0.3 nm of hPD-L1-MMH was first captured onto anti-Penta-His antibodycoated Octet biosensor tips (Fortebio Inc, #18-5079) by submerging thetips for 5 minutes into well containing 20 μg/mL solution of hPD-L1-MMH.The antigen captured biosensor tips were then saturated with firstanti-PD-L1 monoclonal antibody (subsequently referred to as mAb-1) bydipping into wells containing 50 μg/mL solution of mAb-1 for 5 minutes.The biosensor tips were then subsequently dipped into wells containing50 μg/mL solution of a second anti-PD-L1 monoclonal antibody(subsequently referred to as mAb-2). The biosensor tips were washed inOctet HBST buffer in between every step of the experiment. The real-timebinding response was monitored during the course of the experiment andthe binding response at the end of every step was recorded as shown inFIG. 1. The response of mAb-2 binding to hPD-L1-MMH pre-complexed withmAb-1 was compared and competitive/non-competitive behavior of differentanti-PD-L1 monoclonal antibodies was determined.

Under the experimental conditions used in this Example, (a) H2aM8309N,H1H9329P, H1H9336P, H2aM8314N, H2aM8316N, H2aM8718N, H1H9387P2,H1H9351P2, H1H9364P2, H1H9373P2, and H2aM8306N cross-competed with eachother; (b) H2aM8310N, H2aM8321N and H2aM8312N cross-competed with eachother; (c) H1H9396P2, H2aM8317N, H2aM8321N, H1H9323P, H1H9382P2,H1H9344P2, H1H9345P2, and H1H9354P2 cross-competed with each other; and(d) H1H9327P and H2aM8307N cross-competed with each other. In oneinstance, competition was observed in one orientation but not in theopposite orientation: i.e., H2aM8307N when applied first competed withH2aM8309N, H1H9329P, H1H9336P, H2aM8314N, H2aM8316N, H2aM8718N,H1H9387P2, H1H9351P2, H1H9364P2, H1H9373P2, and H2aM8306N; however, inthe opposite orientation, H2aM8309N, H1H9329P, H1H9336P, H2aM8314N,H2aM8316N, H2aM8718N, H1H9387P2, H1H9351P2, H1H9364P2, H1H9373P2, andH2aM8306N when applied first did not compete with H2aM8307N.

Example 7: Antibody Binding to Cells Overexpressing PD-L1

The binding of anti-PD-L1 antibodies to a human embryonic kidney cellline (HEK293; ATCC, #CRL-1573) stably transfected with full length humanPD-L1 (amino acids 1 to 290 of accession number NP_054862.1)(HEK293/hPD-L1) was determined by FACS.

For the assay, adherent cells were detached using enzyme-freedissociation buffer and blocked with complete medium. Cells werecentrifuged and resuspended at a concentration of 2.8×10{circumflex over( )}6 cells/mL in cold PBS containing 2% FBS. HEK293 parental andHEK293/hPD-L1 cells were then incubated for 15 to 30 minutes on ice with100 nM of each anti-PD-L1 antibody or an isotype control antibody.Unbound antibodies were removed by washing with D-PBS containing 2% FBS,and cells were subsequently incubated with a phycoerythrin-conjugatedsecondary Fcγ fragment specifically recognizing either human Fc (JacksonImmunoResearch, #109-116-170) or mouse Fc (Jackson ImmunoResearch,#115-115-164) for 15 to 30 minutes on ice. Cells were washed with D-PBScontaining 2% FBS to remove unbound secondary detection reagents andfluorescence measurements were acquired using a HyperCyte (IntelliCyt,Inc.) flow cytometer. Data was analyzed using HyperCyte software.

TABLE 14 FACS binding of anti-PD-L1 antibodies to HEK293/hPD-L1 cellsand parental HEK293 cells FACS on FACS on Ratio of HEK293 HEK293/HEK293/hPD-L1 parental cells hPD-L1 cells to HEK293 Antibody [MFI] [MFI]parental cells H1H9323P 1909 154992 81 H1H9327P 2120 317592 150 H1H9329P1504 282088 188 H1H9336P 2263 379009 168 H1H9344P2 1691 200976 119H1H9345P2 1885 228406 121 H1H9351P2 1685 289523 172 H1H9354P2 2204275839 125 H1H9364P2 2066 323663 157 H1H9373P2 2151 333236 155 H1H9382P21473 205563 140 H1H9387P2 1232 323793 263 H1H9396P2 2340 227961 97H2aM8306N 1286 316485 246 H2aM8307N 1382 73976 54 H2aM8309N 1160 192678166 H2aM8310N 1357 14918 11 H2aM8312N 1380 158331 115 H2aM8314N 2053194832 95 H2aM8316N 1601 172104 108 H2aM8317N 1270 67600 53 H2aM8321N1322 112495 85 H2aM8323N 2250 163497 73 H2aM8718N 2225 194341 87H2aM8719N 1272 133399 105 mouse IgG 1273 1115 0.9 Isotype control humanIgG1 1179 1099 0.9 Isotype control human IgG4 1991 1839 0.9 Isotypecontrol

As shown in Table 14, all 25 anti-PD-L1 antibodies of the inventionshowed strong binding to the HEK293/hPD-L1 cells compared to binding onthe parental HEK293 line.

Example 8: Blocking of PD-L1-Induced T-Cell Down-Regulation in aT-Cell/APC Luciferase Reporter Assay

T-cell activation is achieved by stimulating T-cell receptors (TcR) thatrecognize specific peptides presented by major histocompatibilitycomplex class I or II proteins on antigen-presenting cells (APC).Activated TcRs in turn initiate a cascade of signaling events that canbe monitored by reporter genes driven by transcription factors such asactivator-protein 1 (AP-1), Nuclear Factor of Activated T-cells (NFAT)or Nuclear factor kappa-light-chain-enhancer of activated B cells(NFκb). T-cell response is modulated via engagement of co-receptorsexpressed either constitutively or inducibly on T-cells. One suchreceptor is programmed cell death protein 1 (PD-1), a negative regulatorof T-cell activity. PD-1 interacts with its ligand, PD-L1, which isexpressed on target cells including APCs or cancer cells, and thisinteraction results in the delivery of inhibitory signals by recruitingphosphatases to the TcR signalosome, resulting in the suppression ofpositive signaling.

A bioassay was developed to measure T cell signaling induced byinteraction between APC and T cells by utilizing a mixed culture derivedfrom two mammalian cell lines: Jurkat cells (an immortalized T cellline) and Raji cells (a B cell line) (FIG. 1). For the first componentof the bioassay, Jurkat Clone E6-1 cells (ATCC, #TIB-152) weretransduced with the Cignal Lenti AP-1 Luc Reporter(Qiagen—Sabiosciences, #CLS-011L) as per the manufacturer'sinstructions. The lentivirus encodes the firefly luciferase gene underthe control of a minimal CMV promoter, tandem repeats of theTPA-inducible transcriptional response element (TRE) and a puromycinresistance gene. The engineered Jurkat cell line was subsequentlytransduced with a PD-1 chimera comprising the extracellular domain ofhuman PD-1 (amino acids from 1 to 170 of human PD1; accession numberNP_005009.2) and the trans-membrane and cytoplasmic domains of humanCD300a (amino acids from 181 to 299 of human CD300a; accession numberNP_009192.2). The resulting stable cell line(Jurkat/AP1-Luc/hPD1-hCD300a) was selected and maintained in RPMI/10%FBS/penicillin/streptomycin/glutamine supplemented with 500 ug/mL G418+1ug/mL puromycin.

For the second component of the bioassay, Raji cells (ATCC, #CCL-86)were transduced with human PD-L1 gene (amino acids 1-290 of accessionnumber NP_054862.1) that had been cloned into a lentiviral (pLEX) vectorsystem (Thermo Scientific Biosystems, #OHS4735). Raji cells, positivefor PD-L1 (Raji/hPD-L1) were isolated by FACS using a PD-L1 antibody andmaintained in Iscove/10% FBS/penicillin/streptomycin/glutaminesupplemented with 1 ug/mL puromycin.

To simulate the APC/T cell interaction, a bispecific antibody composedof one Fab arm that bindings to CD3 on T cells and the other one Fab armbinding that binds to CD20 on Raji cells (CD3×CD20 bispecific antibody;e.g., as disclosed in US20140088295) was utilized. The presence of thebispecific molecule in the assay results in the activation of the T celland APC by bridging the CD3 subunits on T-cells to CD20 endogenouslyexpressed on Raji cells. Ligation of CD3 with anti-CD3 antibodies hasbeen demonstrated to lead to activation of T cells. In this bioassay,antibodies blocking the PD1/PD-L1 interaction rescue T-cell activity bydisabling the inhibitory signaling and subsequently leading to increasedAP1-Luc activation.

In the luciferase-based bioassay, RPMI1640 supplemented with 10% FBS andpenicillin/streptomycin/glutamine was used as assay medium to preparecell suspensions and antibody dilutions to carry out the screening ofanti-PD-L1 monoclonal antibodies (mAbs). On the day of the screening,EC₅₀ values of anti-PD-L1 mAbs, in the presence of a fixed concentrationof CD3×CD20 bispecific antibody (30 pM), as well as the EC₅₀ of thebispecific antibody alone, were determined. In the following order,cells and reagents were added to 96 well white, flat-bottom plates. Forthe anti-PD-L1 mAb EC₅₀ determinations, first a fixed concentration ofCD3×CD20 bispecific antibody (final 30 pM) was prepared and added to themicrotiter plate wells. Twelve-point serial dilutions of anti-PD-L1 mAbsand controls were then added (final concentrations ranging from 1.7 pMto 100 nM; plus wells with assay medium alone). For the bispecificantibody (alone) EC₅₀ determination, the bispecific antibody, at finalconcentrations ranging from 0.17 pM to 10 nM (plus wells with assaymedium alone), was added to the microtiter plate wells. Subsequently, a2.5×10{circumflex over ( )}6/mL Raji/hPD-L1 cell suspension was preparedand 20 uL per well was added (final cell number/well 5×10{circumflexover ( )}4 cells). Plates were left at room temperature (15-20 minutes),while a suspension of 2.5×10{circumflex over ( )}6/mL ofJurkat/AP1-Luc/hPD1(ecto)-hCD300a(TM-Cyto) was prepared. 20 uL of theJurkat suspension (final cell number/well 5×10{circumflex over ( )}4cells) was added per well. Plates containing the co-culture wereincubated for 5 to 6 hours at 37° C. in 5% CO₂. Luciferase activity wasthen detected after the addition of ONE-Glo™ (Promega, #E6051) reagentand relative light units (RLUs) were measured on a Victor luminometer.All samples were tested in duplicates.

RLU values for each screened antibody were normalized by setting theassay condition with fixed (30 pM) concentration of the CD3/CD20bispecific antibody, but without anti-PD-L1 antibody to 100%. Thiscondition corresponds to the maximal AP1-Luc response elicited by thebispecific molecule in the presence of the PD-1/PD-L1 inhibitory signal.Upon addition of the anti-PD-L1 antibody, the inhibitory signal issuppressed, and the increased stimulation is shown here as E_(max), thepercentage increase in the signal in the presence of the highestantibody dose tested (100 nM). To compare potency of the anti-PD-L1antibodies tested, the concentration of antibody at which the normalizedRLU value reached 125% activation was determined from a four-parameterlogistic equation over a 12-point response curve using GraphPad Prism.The results are summarized in Table 15.

TABLE 15 Anti-PD-L1 antibody blocking PD-1/PD-L1 dependent inhibition ofAP1-Luc signaling Antagonistic assay Antagonistic assay Concentration[M] of Emax mean Antibody at 125% [%] @ Antibody activation 100 nMH2aM8306N 3.78E−09 166.8 H2aM8307N N/A 87.7 H2aM8309N 3.09E−10 180.9H2aM8310N N/A 112.8 H2aM8312N N/A 75.6 H2aM8314N 1.44E−11 234.9H2aM8316N 1.47E−10 177.4 H2aM8317N N/A 109.2 H2aM8321N N/A 116.8H2aM8323N 2.20E−10 173.3 H2aM8718N 1.51E−10 182.0 H1H9323P N/A 101.1H1H9327P N/A 77.1 H1H9329P N/A 124.5 H1H9336P 9.81E−11 162.6 H1H9344P2N/A 98.3 H1H9345P2 N/A 89.2 H1H9351P2 3.44E−10 154.4 H1H9354P2 N/A 89.9H1H9364P2 7.93E−11 164.5 H1H9373P2 1.34E−10 150.2 H1H9382P2 N/A 86.5H1H9387P2 1.86E−11 141.2 H1H9396P2 N/A 102.6 H1H8314N 6.57E−11 147.9H1H9364P2 1.62E−10 158.1 H1H9373P2 7.07E−11 152.5 mlgG2a isotype controlN/A 80.2 hlgG1 isotype control N/A 96.8 hlgG4 isotype control N/A 87.6N/A = not applicable because at the concentrations tested theseantibodies did not activate 125%

As shown in Table 15, 14 out of the 27 anti-PD-L1 antibodies of theinvention tested blocked PD-1/PD-L1 inhibition with E_(max) valuesranging from 234.9 to 138.1. Thirteen out of the 27 anti-PD-L1antibodies of the invention did not demonstrate substantial blockade ofPD1/PD-L1 interaction when tested in this assay. Isotype controls didnot interfere with the PD1/PD-L1 interaction.

Example 9: In Vivo Efficacy of Anti-PD-L1 Antibodies

To determine the effect of a select number of anti-PD-L1 antibodies ofthe invention in a relevant in vivo model, an MC38.ova tumor growthstudy, involving subcutaneous injection of tumor cells and started ondifferent days, was conducted in mice that were homozygous for theexpression of the extracellular domain of human PD-L1 in place ofextracellular domain of mouse PD-L1 (PD-L1 HumIn mice) on a 75%C57/BI6/25% 129 strain background. MC38.Ova (mouse colon adenocarcinoma)cells were engineered to express chicken ovalbumin in order to increasetumor immunogenicity, and to allow monitoring of the T-cell immuneresponses to well-defined antigenic ovalbumin peptides. In a secondstep, MC38.Ova cells were transduced with a lentiviral vector expressinghPD-L1 under SFFV promoter. MC38.Ova cells positive for hPD-L1(MC38.Ova/hPD-L1) were isolated by FACS using hPD-L1-specific antibody.The cells were found to express low level of endogenous mouse PD-L1.

For a first study (study #1), mice were divided evenly according to bodyweight into 5 treatment or control groups (n=5 to 8 mice per group). Atday 0, mice were anesthetized by isoflurane inhalation and then injectedsubcutaneously into the right flank with 1×10⁶ MC38.ova/hPD-L1 cells insuspension of 100 uL of DMEM. Treatment groups were intraperitoneallyinjected with 500 ug of either one of three anti-PD-L1 antibodies of theinvention, or one of two isotype control antibodies with irrelevantspecificity on days 3, 7, 10, 14, and 17 of the experiment, while onegroup of mice was left untreated.

In a second study (study #2), PD-L1 humanized mice were randomized into7 treatment groups (n=5 to 6 mice). On day 0, mice were subcutaneouslyimplanted with 1×10{circumflex over ( )}6 MC38.Ova/hPD-L1 cells. Micewere intraperitoneally administered with REGN a-PD-L1 ab (H1H8314N orH1H9364P2 or H1H9373P2), or isotype control abs hIgG4 mut or hIgG1 atdoses of 10 mg/kg or 5 mg/kg. Groups of mice were administered antibodyon days 3, 7, 10, 14, and 17. Tumor volumes were monitored by calipermeasurement twice per week for the duration of the experiment (21 days).Experimental dosing and treatment protocol for groups of mice are shownin Table 16.

TABLE 16 Experimental dosing and treatment protocol for groups of miceSamples Dosage amount at each Study # tested dosage time point Dosinginterval 1 Isotype control 1 500 μg Days 3, 7, 10, 14, 17 H1H8314N 500μg Days 3, 7, 10, 14, 17 H1H9364P2 500 μg Days 3, 7, 10, 14, 17H1H9373P2 500 μg Days 3, 7, 10, 14, 17 Isotype control 2 500 μg Days 3,7, 10, 14, 17 2 Isotype control 1 10 mg/kg Days 3,7, 10, 14, 17 H1H8314N10 mg/kg Days 3, 7, 10, 14, 17 H1H8314N 5 mg/kg Days 3, 7, 10, 14, 17H1H9364P2 10 mg/kg Days 3, 7, 10, 14, 17 H1H9364P2 5 mg/kg Days 3, 7,10, 14, 17 H1H9373P2 10 mg/kg Days 3, 7, 10, 14, 17 H1H9373P2 5 mg/kgDays 3, 7, 10, 14, 17

For the studies, average tumor volumes were monitored by calipermeasurement twice per week for the duration of the experiment (17 days)and percent survival was recorded at the end of the experiment. Inaddition, the number of tumor-free mice was also assessed at the end ofthe study. Results, expressed as mean tumor volume (mm³)(±SD), percentsurvival, and number of tumor-free mice are shown in Tables 17 and 18.

TABLE 17 Mean tumor volume, percent survival and numbers of tumor-freemice in each treatment group from study #1 Tumor Volume, mm³ Tumor-Freemean (±SD) Survival, % Mice Antibody Days 10 Day 17 Day 10 Day 17 Day 17Isotype Control 1 65 (±27) 148 (±109) 100% 100% 0/5 (0%) Isotype Control2 54 (±44) 80 (±63) 100% 100% 0/5 (0%) H1H8314N  6 (±10) 2 (±5) 100%100%  4/5 (80%) H1H9364P2 16 (±17) 0 (±0) 100% 100%   5/5 (100%)H1H9373P2 13 (±14) 0 (±0) 100% 100%   5/5 (100%)

As shown in Table 17 for study #1, all three anti-PD-L1 antibodies ofthe invention were efficacious in promoting tumor regression at thedosage of 500 ug/mouse with all mice from treatment groups that receivedtwo of the antibodies, H1H9364P2 and H1H9373P2, being tumor free at day17. In the treatment group that received one of the anti-PD-L1antibodies of the invention, H1H8314N, 4 out of 5 mice were tumor freeby day 17, whereas 0 out of 5 animals were tumor-free in the isotypecontrol groups. One-way ANOVA with Dunnett's multiple comparisonpost-test revealed a significant difference in tumor volumes betweentreatments with anti-PD-L1 antibodies of the invention and the isotypecontrol antibody with a p value <0.05.

TABLE 18 Mean tumor volume, percent survival and numbers of tumor-freemice in each treatment group from study #2 Tumor Volume, mm3 Tumor-Tumor- Mean (SD) Survival, % free mice free mice Day 10 Day 10 Day 21Day 21 Day 10 Day 10 Day 21 Day 21 Day 21 Day 21 Antibody 10 MPK 5 MPK10 MPK 5 MPK 10 MPK 5 MPK 10 MPK 5 MPK 10 MPK 5 MPK Isotype 55 (37) N/A534 N/A 100% N/A 100% N/A 0/6 (0%) N/A control 1 (356) H1H8314N 14 (15)17 (4)  19 108 100% 100% 100% 100% 3/6 2/5 (40%) 922) (101) (50%)H1H9364P2 18 (10) 23 (10) 34 (81) 231 100% 100% 100% 100% 5/6 1/5 (20%)(238) (83%) H1H9373P2 10 (8)  25 929) 7 (16) 37 (59) 100% 100% 100% 100%5/6 3/5 (60%) (83%)

As shown in Table 18, in study #2, administration of the selectedanti-PD-L1 antibodies resulted in inhibition of tumor growth promotingtumor regression. All three anti-PD-L1 antibodies were efficacious atthe 10 mg/kg dose and 5 mg/kg dose and promoted tumor regression intreated mice in a dose dependent manner throughout the course of theexperiment, whereas 0 out of 5 animals were tumor-free in the controlgroup. One-way ANOVA with Tukey's multiple comparison post-test revealeda significant difference in tumor volumes between treatments with theanti-PD-L1 antibodies and isotype control antibody with p value <0.05 orlower.

Example 10: Anti-Tumor Effects of a Combination of an Anti-PD-L1Antibody and a VEGF Antagonist in a Mouse Early-Treatment Tumor Model

An early-treatment tumor model was developed to test the efficacy of acombination of an anti-PD-L1 antibody and a VEGF antagonist. In thismodel, the combination therapy is administered shortly after tumorimplantation. The experiment also used an anti-PD-1 antibody alone andin combination with the VEGF antagonist. The anti-PD-L1 antibody used inthis experiment was an anti-PD-L1 monoclonal antibody with V_(H)/V_(L)sequences of antibody “YW243.55S70” according to US20100203056A1(Genentech, Inc.), with mouse IgG2a and which was cross-reactive withmouse PD-L1. The VEGF antagonist used in this experiment was aflibercept(a VEGF receptor-based chimeric molecule, also known as “VEGF-trap” or“VEGFR1R2-FcΔC1(a),” a full description of which is provided elsewhereherein). The anti-PD-1 antibody used in this experiment was anti-mousePD-1 clone “RPMI-14” with rat IgG2b (Bio X Cell, West Lebanon, N.H.).

For this experimental model, 1.0×10⁶ Colon-26 tumor cells were implantedsub-cutaneously into BALB/c mice at Day 0. Starting on Day 3, prior tothe establishment of measurable tumors, mice were treated with one ofthe mono- or combination therapies, or control combination, as set forthin Table 19.

TABLE 19 Experimental Dosing and Treatment Groups Treatment Group FirstAgent Second Agent Control IgG2a isotype hFc control Combination control(250 μg, IP) (250 μg, SC) VEGF Trap IgG2a isotype Aflibercept onlycontrol (250 μg, IP) (10 mg/kg, SC) anti-PD-1 anti-PD-1 mAb hFc controlonly RPMI-14 (250 μg, IP) (250 μg, SC) anti-PD-L1 anti-PD-L1 mAb hFccontrol only (250 μg, IP) (250 μg, SC) VEGF Trap + anti-PD-1 mAbAflibercept anti-PD-1 RPMI-14 (250 μg, IP) (10 mg/kg, SC) VEGF Trap +anti-PD-L1 mAb Aflibercept anti-PD-L1 (250 μg, IP) (10 mg/kg, SC)

The various therapies were administered at five different time pointsover a two week period (i.e., injections at Day 3, Day 6, Day 10, Day 13and Day 19).

Animals in each therapy group were evaluated in terms of tumorincidence, tumor volume, median survival time, and number of tumor-freeanimals at Day 50. The extent of tumor growth is summarized in FIG. 2(tumor growth curves) and FIG. 3 (tumor volume at Day 28). Results arealso summarized in Table 20.

TABLE 20 Tumor-free mice upon treatment No. of Tumor-Free TreatmentGroup Animals by Day 50 Control Combination 0/10 VEGF Trap only 3/10anti-PD-1 only 4/10 anti-PD-L1 only 5/10 VEGF Trap + anti-PD-1 7/10 VEGFTrap + anti-PD-L1 9/10

Tumor growth was substantially reduced in animals treated with thecombination of VEGF Trap+ anti-PD-L1 antibody as compared with treatmentregimens involving either therapeutic agent alone (see FIGS. 2 and 3).Furthermore, survival was substantially increased in the VEGF Trap+anti-PD-L1 antibody group, with 90% of animals surviving to at least day50 after tumor implantation. By contrast, for the anti-PD-L1 and VEGFTrap monotherapy groups, survival to Day 50 was only 50% and 30%respectively (see FIG. 3 and Table 20).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that competes for binding to human programmed death-ligand 1(PD-L1) protein with a reference antibody comprising the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR),wherein the HCVR has an amino acid sequence selected from the groupconsisting of HCVR sequences listed in Table 1; and the CDRs of a lightchain variable region (LCVR), wherein the LCVR has an amino acidsequence selected from the group consisting of LCVR sequences listed inTable 1 and wherein the antibody has one or more of the followingproperties: (a) binds monomeric PD-L1 with a binding dissociationequilibrium constant (K_(D)) of less than about 310 pM as measured in asurface plasmon resonance assay at 37° C.; (b) binds monomeric humanPD-L1 with a K_(D) less than about 180 pM in a surface plasmon resonanceassay at 25° C.; (c) binds dimeric human PD-L1 with a K_(D) of less thanabout 15 pM as measured in a surface plasmon resonance assay at 37° C.;and (d) binds dimeric human PD-L1 with a K_(D) less than about 8 pM in asurface plasmon resonance assay at 25° C.
 2. The isolated antibody orantigen-binding fragment of claim 1, wherein the reference antibody orantigen-binding fragment thereof comprises an HCVR/LCVR amino acidsequence pair as set forth in Table
 1. 3. The isolated antibody orantigen-binding fragment of claim 2, wherein the reference antibodycomprises an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 82/90, 98/106, 146/154, 162/170, 290/274,306/274, 314/274 and 330/274.
 4. An isolated human monoclonal antibodyor antigen-binding fragment thereof that binds specifically to humanPD-L1, wherein the antibody or antigen-binding fragment thereofcomprises a HCVR having an amino acid sequence selected from the groupconsisting of HCVR sequences listed in Table
 1. 5. An isolated humanmonoclonal antibody or antigen-binding fragment thereof that bindsspecifically to human PD-L1, wherein the antibody or antigen-bindingfragment thereof comprises a LCVR having an amino acid sequence selectedfrom the group consisting of LCVR sequences listed in Table
 1. 6. Anisolated human monoclonal antibody or antigen-binding fragment thereofthat binds specifically to human PD-L1, wherein the antibody orantigen-binding fragment thereof comprises (a) a HCVR having an aminoacid sequence selected from the group consisting of HCVR sequenceslisted in Table 1; and (b) a LCVR having an amino acid sequence selectedfrom the group consisting of LCVR sequences listed in Table
 1. 7. Theisolated antibody or antigen-binding fragment thereof of claim 6,wherein the antibody or antigen-binding fragment thereof comprises threeheavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one ofthe HCVR sequences listed in Table 1; and three light chain CDRs (LCDR1,LCDR2 and LCDR3) contained within any one of the LCVR sequences listedin Table
 1. 8. The isolated antibody or antigen-binding fragment thereofof claim 7, comprising: (a) a HCDR1 domain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84,100, 116, 132, 148, 164, 180, 188, 204, 220, 236, 252, 268, 284, 292,300, 308, 316, 324, 332, and 340; (b) a HCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 22,38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 190, 206, 222, 238, 254,270, 286, 294, 302, 310, 318, 326, 334, and 342; (c) a HCDR3 domainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 192, 208,224, 240, 256, 272, 288, 296, 304, 312, 320, 328, 336, and 344; (d) aLCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156,172, 196, 212, 228, 244, 260, and 276; (e) a LCDR2 domain having anamino acid sequence selected from the group consisting of SEQ ID NOs:14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 198, 214, 230, 246,262, and 278; and (f) a LCDR3 domain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80,96, 112, 128, 144, 160, 176, 200, 216, 232, 248, 264, and
 280. 9. Theisolated antibody or antigen-binding fragment of claim 8, comprising aHCVR/LCVR amino acid sequence pair selected from the group consisting ofSEQ ID NOs: 82/90, 98/106, 146/154, 162/170, 290/274, 306/274, 314/274and 330/274.
 10. An isolated antibody or antigen-binding fragmentthereof that blocks PD-L1 binding to one of PD-1 or B7-1, the isolatedantibody or antigen-binding fragment thereof comprising the CDRs of aHCVR, wherein the HCVR has an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 34, 50, 82, 98, 146, 162, 178, 186,234, 250, 266, 290, 306, 314, and 330; and the CDRs of a LCVR, whereinthe LCVR has an amino acid sequence selected from the group consistingof SEQ ID NOs: 10, 42, 58, 90, 106, 154, 170, 194, 242, 258, and 274.11. An isolated antibody or antigen-binding fragment thereof that bindsPD-L1, wherein the antibody or antigen-binding fragment thereof enhancesPD-L1 binding to one of PD-1 or B7-1.
 12. The isolated antibody orantigen-binding fragment thereof of claim 11, wherein the antibodycomprises the CDRs of a HCVR, wherein the HCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 18, 66, 114,130, 202, 218, 282, 298, 322, and 338; and the CDRs of a LCVR, whereinthe LCVR has an amino acid sequence selected from the group consistingof SEQ ID NOs: 26, 74, 122, 138, 210, 226, and
 274. 13. The isolatedantibody or antigen-binding fragment thereof of claim 11 or 12, whereinthe antibody comprises an HCVR/LCVR amino acid sequence pair selectedfrom the group consisting of SEQ ID NOs: 18/26, 66/74, 114/122, 130/138,202/210, 218/226, 282/274, 298/274, 322/274, and 338/274.
 14. Theantibody or antigen-binding fragment thereof of any one of claims 1-13,wherein the antibody is a multi-specific antigen-binding molecule.
 15. Apharmaceutical composition comprising an isolated antibody orantigen-binding fragment thereof that binds to PD-L1 according to anyone of claims 1-14 and a pharmaceutically acceptable carrier or diluent.16. An isolated polynucleotide molecule comprising a polynucleotidesequence that encodes a HCVR of an antibody as set forth in any one ofclaims 1-7 or 9-14.
 17. An isolated polynucleotide molecule comprising apolynucleotide sequence that encodes a LCVR of an antibody as set forthin any one of claims 1-6 or 8-14.
 18. A vector comprising thepolynucleotide sequence of claim 16 or
 17. 19. A cell expressing thevector of claim
 18. 20. A multi-specific antigen-binding molecule orfragment thereof comprising a first antigen-binding specificity thatbinds specifically to PD-L1 and a second antigen-binding specificitythat binds specifically to an antigen selected from the group consistingof a tumor-cell-specific antigen, an antigen specific to avirally-infected cell, and a T-cell co-inhibitor.
 21. The multi-specificantigen-binding molecule or fragment thereof of claim 20, wherein thefirst antigen-binding specificity comprises three heavy chaincomplementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3)contained within any one of the heavy chain variable region (HCVR)sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2and LCDR3) contained within any one of the light chain variable region(LCVR) sequences listed in Table
 1. 22. The multi-specificantigen-binding molecule or fragment thereof of claim 21, wherein thefirst antigen-binding specificity comprises a HCVR having an amino acidsequence selected from the group consisting of HCVR sequences listed inTable 1; and a LCVR having an amino acid sequence selected from thegroup consisting of LCVR sequences listed in Table
 1. 23. Themulti-specific antigen-binding molecule or fragment thereof of claim 22,wherein the first antigen-binding specificity comprises a HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 82/90, 98/106, 146/154, 162/170, 290/274, 306/274, 314/274 and330/274.
 24. The multi-specific antigen-binding molecule or fragmentthereof of claim 20, wherein the first antigen-binding specificitycomprises an extracellular domain of one of PD-1 or B7-1, or fragmentthereof.
 25. The multi-specific antigen-binding molecule or fragmentthereof of claim 20, wherein the second antigen-binding specificitybinds specifically to a T-cell co-inhibitor selected from the groupconsisting of LAG3, TIM3, B7-1, CTLA-4, BTLA, CD28, 2B4, LY108, TIGIT,ICOS, and CD160.
 26. The multi-specific antigen-binding molecule orfragment thereof of any one of claims 20-24, wherein the secondantigen-binding specificity binds specifically to a tumor cell-specificantigen selected from the group consisting of CA9, CA125,melanoma-associated antigen (MAGE), carcinoembryonic antigen (CEA),vimentin, tumor-M2-PK, prostate-specific antigen (PSA), MART-1, andCA19-9.
 27. The multi-specific antigen-binding molecule or fragmentthereof of any one of claims 20-24, wherein the second antigen-bindingspecificity binds specifically to an antigen from a cell infected with avirus selected from the group consisting of human immunodeficiency virus(HIV), hepatitis C virus (HCV), human papilloma virus (HPV), lymphocyticchoriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).28. The multi-specific antigen-binding molecule or fragment thereof ofany one of claims 20-26 for use in the treatment of a cancer selectedfrom the group consisting of renal cell carcinoma, colorectal cancer,ovarian cancer, prostate cancer, breast cancer, colon cancer,non-small-cell lung cancer and melanoma.
 29. The multi-specificantigen-binding molecule or fragment thereof any one of claim 20-25 or27 for use in the treatment of a chronic viral infection caused by avirus selected from the group consisting of HIV, HPV, HBV, HCV, LCMV andSIV.
 30. A multi-specific antigen-binding molecule or antigen-bindingfragment thereof comprising a first binding specificity that bindsspecifically to PD-L1 and a second binding specificity that comprises anextracellular domain of PD-1 or B7-1, or fragment thereof.
 31. Themulti-specific antigen-binding molecule or fragment thereof of claim 30for use in the treatment of a cancer selected from the group consistingof renal cell carcinoma, colorectal cancer, ovarian cancer, prostatecancer, breast cancer, colon cancer, non-small-cell lung cancer andmelanoma.
 32. The multi-specific antigen-binding molecule or fragmentthereof of claim 30 for use in the treatment of a chronic viralinfection caused by a virus selected from the group consisting of HIV,HPV, HBV, HCV, LCMV and SIV.
 33. A method of enhancing an immuneresponse in a subject, the method comprising administering apharmaceutical composition comprising an isolated antibody orantigen-binding fragment thereof according to any one of claims 1-14; ora multi-specific antigen-binding molecule or fragment thereof accordingto any one of claim 20-27 or
 30. 34. A method of inhibiting aT-regulatory (Treg) cell in a subject comprising administering apharmaceutical composition comprising an isolated human antibody orantigen-binding fragment thereof according to any one of claims 1-14; ora multi-specific antigen-binding molecule or fragment thereof accordingto any one of claim 20-27 or
 30. 35. A method of enhancing T-cellactivation in a subject, the method comprising administering apharmaceutical composition comprising an antibody or antigen-bindingfragment thereof according to any one of claims 1-14; or amulti-specific antigen-binding molecule or fragment thereof according toany one of claim 20-27 or
 30. 36. The method of any one of claims 33-35,wherein the subject has a disease or disorder selected from the groupconsisting of renal cell carcinoma, ovarian cancer, prostate cancer,non-small-cell lung cancer, colorectal cancer, and melanoma.
 37. Themethod of any one of claims 33-35, wherein the subject has a chronicviral infection caused by a virus selected from the group consisting ofHIV, HCV, HBV, HPV, LCMV and SIV.
 38. A method of inhibiting growth of atumor or a tumor cell, the method comprising contacting the tumor ortumor cell with a therapeutically effective amount of the antibody ofany one of claim 1-14 or 20-26.
 39. The method of any one of claims33-38, wherein the antibody or antigen-binding fragment thereof, or thepharmaceutical composition comprising the antibody or antigen-bindingfragment thereof, is administered to the subject in combination with asecond therapeutic agent.
 40. The method of claim 39, wherein the secondtherapeutic agent is selected from the group consisting of a NSAID, acorticosteroid, an antibody to a T-cell co-inhibitor, an antibody to atumor specific antigen, an antibody to an autoimmune tissue antigen, anantibody to a virally-infected-cell antigen, an antibody to PD-1, adietary supplement such an antioxidant, a VEGF antagonist, achemotherapeutic agent, a cytotoxic agent, and an anti-viral drug. 41.The method of any one of claims 33-40, wherein the antibody orantigen-binding fragment thereof is administered subcutaneously,intravenously, intradermally, intraperitoneally, orally, intramuscularlyor intracranially.
 42. The method of any one of claims 33-41, whereinthe antibody or antigen-binding fragment is administered at a dose ofabout 0.1 mg/kg of body weight to about 60 mg/kg of body weight of thesubject.